Influence of follicular fluid meiosis-activating sterol on aneuploidy rate and precocious chromatid segregation in aged mouse oocytes.

BACKGROUND: Follicular fluid meiosis-activating sterol (FF-MAS) protects young oocytes from precocious chromatid separation (predivision). Reduced expression of cohesion and checkpoint proteins and predivision has been hypothesized to occur in age-related aneuploidy in oocytes. METHODS: To know whether FF-MAS also protects aged oocytes from predivision and from age-related non-disjunction, we analysed chromosome constitution in mouse oocytes matured spontaneously with or without 10 microM FF-MAS and in hypoxanthine (HX)-arrested young and aged oocytes induced to resume maturation by FF-MAS. Messenger RNA for checkpoint protein MAD2 and cohesion protein SMC1beta was compared between oocytes matured with or without FF-MAS. RESULTS: Aged oocytes possessed many bivalents with single distal chiasma at meiosis I. Predivision was especially high in aged oocytes cultured sub-optimally to metaphase II in alpha-minimum essential medium (alpha-MEM). FF-MAS reduced predivision significantly (P < 0.001) but neither reduced non-disjunction nor induced aneuploidy in aged oocytes. Polyploidy was high in FF-MAS-stimulated maturation, in particular in the aged oocytes (P > 0.001). Relative levels of Smc1beta mRNA appeared increased by maturation in FF-MAS, and mitochondrial clustering was restored. CONCLUSIONS: Sister chromatids of aged oocytes appear to be highly susceptible to precocious chromatid separation, especially when maturation is under sub-optimal conditions, e.g. in the absence of cumulus and FF-MAS. This may relate to some loss of chromatid cohesion during ageing. FF-MAS protects aged oocytes from predivision during maturation, possibly by supporting Smc1beta expression, thus reducing risks of meiotic errors, but it cannot prevent age-related non-disjunction. Aged oocytes appear prone to loss of co-ordination between nuclear maturation and cytokinesis suggesting age-related relaxed cell cycle control.

Functional effects and molecular mechanisms of subtype-selective ERalpha and ERbeta agonists in the cardiovascular system.

Gender differences in the development of cardiovascular disease suggested for a protective function of estrogens in heart disease. The negative or neutral outcome of clinical trials on hormone replacement therapy provides clear evidence that the role of female sex hormones in the cardiovascular system is more complex than previously thought. In particular, the function of estrogens can not be understood without detailed knowledge on the specific function of both estrogen receptor subtypes in the heart and in the vasculature. In here, we review recent studies on subtype selective ERalpha and ERbeta agonists in different animal models of hypertension, cardiac hypertrophy and vascular inflammation. The results indicate that the activation of specific ER subtypes confers specific as well as redundant protective effects in hypertensive heart disease that might ultimately translate into novel treatment options for hypertensive heart disease.

Meiosis-activating sterol protects oocytes from precocious chromosome segregation.

BACKGROUND: Follicular fluid meiosis-activating sterol (FF-MAS) overcomes hypoxanthine (HX)-mediated meiotic arrest in mammalian oocytes. METHODS: In order to determine whether chromosome segregation was normal in oocytes matured in FF-MAS, the development, chromosomal constitution and chromosome alignment was analysed in spontaneously matured as well as HX-arrested mouse oocytes cultured in the absence or presence of FF-MAS. RESULTS: FF-MAS-induced meiotic maturation was significantly less effective compared with spontaneous maturation in supporting cytokinesis ( approximately 40 and approximately 90% polar body formation respectively). The majority of oocytes stimulated by FF-MAS to overcome the HX block developed to metaphase II (MII), but 23.4% of meiosis II oocytes were diploid. Chromosomes were well aligned on the spindle, and hyperploidy was low in spontaneously matured oocytes and HX-arrested oocytes cultured with or without FF-MAS. Unexpectedly, almost 40% of spontaneously matured MII oocytes contained chromatids/monads. Precocious loss of chromatid cohesion was significantly reduced in spontaneously matured as well as HX-arrested oocytes cultured in the presence of FF-MAS but not lanosterol. CONCLUSIONS: FF-MAS induces full nuclear maturation to MII, and chromosomes segregate with high fidelity. However, in delayed FF-MAS-stimulated meiotic maturation, anaphase I may occur in the absence of cytokinesis. FF-MAS appears to protect mammalian oocytes from precocious chromatid segregation.

Activation of meiotic maturation in rat oocytes after treatment with follicular fluid meiosis-activating sterol in vitro and ex vivo.

Meiosis-activating sterols (MAS) have been found to induce meiotic maturation in mouse oocytes in vitro. In the present study we have extended these observations by investigating the effects of follicular fluid MAS (FF-MAS) on rat oocyte maturation in vitro and ex vivo. Rat oocytes freed from their follicles were cultured with FF-MAS (0 microM, 1 microM, 3 microM, 10 microM, 30 microM) for 22 h in a medium containing the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX; 250 microM). A dose-dependent significant increase in germinal vesicle breakdown (GVB) was observed after adding FF-MAS to the culture medium in both cumulus-enclosed (CEO) and denuded (DO) oocytes. A time course study (0, 3, 8, 14, and 22 h) showed a significant increase in GVB after 14 h when DO and CEO were cultured in the presence of 10 microM FF-MAS + 250 microM IBMX. Furthermore immature rats were primed with eCG (20 IU) and 48 h later perfused ex vivo for 12 h in a recirculating system with either FF-MAS (0 microM, 10 microM, 30 microM, 60 microM), cholesterol (60 microM), or LH (0.2 microg/ml) in the presence of 200 microM IBMX, respectively. In addition, ovarian perfusion was carried out with FF-MAS (30 microM, 60 microM) or 0.2 microg/ml LH in the absence of IBMX. After 12 h, oocytes were freed from the ovaries and checked for GVB. By using the ex vivo perfused rat ovary, we found that FF-MAS, starting at 30 microM, was dose-dependently able to overcome IBMX-induced meiotic arrest leading to a comparable increase in GVB as was observed for LH. Furthermore, it was found that FF-MAS in the absence of IBMX was also able to induce meiotic maturation. Our data are consistent with the notion that the maturation-inducing effects of FF-MAS are mediated by different mechanisms compared to spontaneous maturation.

Modulation of endometrial transformation in gonadotrophin-stimulated and unstimulated pseudo-pregnant rabbits: studies with the progesterone receptor antagonist, onapristone.

Advanced endometrial transformation often occurs in IVF and embryo transfer therapy after ovarian stimulation with gonadotrophins. One reason is probably the early rise in peripheral progesterone concentration after ovulation induction. Consequently, we studied in a rabbit model, whether the post-ovulatory application of the progesterone receptor antagonist, onapristone, could prevent such an advancement of endometrial transformation after stimulation with different gonadotrophin preparations. The inhibitory effect of onapristone on the endometrium is dependent upon the strength of ovarian stimulation. In unstimulated animals or animals treated with recombinant LH (nine corpora lutea/animal in both groups), secretory differentiation and proliferation of the endometrium was strongly inhibited by onapristone. After weak ovarian stimulation with a 3:1 mixture of FSH and LH (22 corpora lutea/animal), secretory differentiation was strongly inhibited, while proliferation was enhanced. After strong stimulation with either a 1:1 mixture of FSH and LH, or human menopausal gonadotrophin (HMG; >40 corpora lutea/animal), only limited inhibitory effects of onapristone on secretory transformation or proliferation could be detected. In conclusion, these graded effects of onapristone after stimulation with gonadotrophins, resemble the basic observations from which a therapeutic strategy emerges, to modulate the advanced endometrial transformation which occurs in many IVF patients after ovarian stimulation.

Luteinizing hormone induces mouse vas deferens protein expression in the murine ovary.

The aim of our study was to isolate and identify novel proteins that are involved in the process of ovulation. To achieve this goal we used the technique of proteome analysis. Comparison of ovary protein patterns, obtained by high resolution two-dimensional gel electrophoresis from recombinant FSH (rFSH)- and rFSH + human CG (hCG)-treated mice, showed significant differences in protein spot positions and intensities. Subsequent analysis of one of these proteins was performed by mass spectrometry, resulting in the identification of the mouse vas deferens protein (MVDP). MVDP, which was absent in the two-dimensional gel electrophoresis protein pattern of rFSH-primed mice and appeared 3 h after the hCG surge, is a member of the aldo-keto reductase superfamily and was originally identified in the mouse vas deferens. This is the first study describing MVDP expression and regulation by LH in the ovary. Northern blot analysis of female mice tissues showed that mvdp messenger RNA (mRNA) was only present in adrenal glands and in hCG-treated ovaries. In situ hybridization studies localized the mvdp mRNA unequivocally to ovarian thecal and interstitial cells with an expression profile starting already 1.5 h, and decreasing 24 h, after LH treatment. In the adrenal glands, mvdp mRNA was not regulated by LH and localized in the cells of the zona fasciculata. In murine adrenocortical cells, a recent study proposed a detoxifying role of MVDP. MVDP might fulfill the same function in the ovary; however, because of its strong and early transcriptional induction by LH, it is also possible that MVDP catalyses another important step during the cascade of events occurring at the time of ovulation.

Identification of selective estrogen receptor modulators by their gene expression fingerprints.

Clinical studies have shown that estrogen replacement therapy (ERT) reduces the incidence and severity of osteoporosis and cardiovascular disease in postmenopausal women. However, long term estrogen treatment also increases the risk of endometrial and breast cancer. The selective estrogen receptor (ER) modulators (SERMs) tamoxifen and raloxifene, cause antagonistic and agonistic responses when bound to the ER. Their predominantly antagonistic actions in the mammary gland form the rationale for their therapeutic utility in estrogen-responsive breast cancer, while their agonistic estrogen-like effects in bone and the cardiovascular system make them candidates for ERT regimens. Of these two SERMs, raloxifene is preferred because it has markedly less uterine-stimulatory activity than either estrogen or tamoxifen. To identify additional SERMs, a method to classify compounds based on differential gene expression modulation was developed. By analysis of 24 different combinations of genes and cells, a selected set of assays that permitted discrimination between estrogen, tamoxifen, raloxifene, and the pure ER antagonist ICI164384 was generated. This assay panel was employed to measure the activity of 38 compounds, and the gene expression fingerprints (GEFs) obtained for each compound were used to classify all compounds into eight groups. The compound's GEF predicted its uterine-stimulatory activity. One group of compounds was evaluated for activity in attenuating bone loss in ovariectomized rats. Most compounds with similar GEFs had similar in vivo activities, thereby suggesting that GEF-based screens could be useful in predicting a compound's in vivo pharmacological profile.

Meiosis-activating sterol-mediated resumption of meiosis in mouse oocytes in vitro is influenced by protein synthesis inhibition and cholera toxin.

To explore the possible signaling pathways of meiosis-activating sterol (MAS)-induced oocyte maturation and to elucidate whether the MAS pathway involves transcription or translation, arrested immature mouse oocytes were cultured with either the protein synthesis inhibitor cycloheximide or the heteronuclear RNA inhibitors alpha-amanitin or actinomycin D, respectively. Moreover, the possible involvement of a G protein-coupled receptor mechanism in MAS-mediated oocyte maturation was explored by influencing oocyte maturation with cholera toxin (CT). MAS-induced oocyte maturation was completely blocked by the addition of 50 microg/ml cycloheximide 4 h before the addition of MAS. Simultaneous addition of MAS and the protein synthesis inhibitor also significantly reduced the meiotic resumption compared to that in MAS-treated controls. In contrast, neither of the treatment regimens to inhibit transcription of DNA to RNA was observed to have any effect on the MAS-induced resumption of meiosis. CT was observed to inhibit MAS-induced, but not spontaneous, oocyte maturation in vitro, suggesting a putative involvement of G protein-coupled receptor mechanism in the MAS mode of action. In conclusion, protein synthesis was found to be an essential requirement for maintaining the oocytes' responsiveness to MAS-induced resumption of meiosis, in contrast to transcription.

Effects of tamoxifen on serotonin transporter and 5-hydroxytryptamine(2A) receptor binding sites and mRNA levels in the brain of ovariectomized rats with or without acute estradiol replacement.

Estradiol-17beta (E(2)), in its positive feedback mode for gonadotropin release in the female rat, induces expression of the genes for the 5-hydroxytryptamine(2A) receptor (5-HT(2A)R) and the serotonin transporter (SERT) in the dorsal raphe nucleus (DRN) with a concomitant increase in the densities of 5-HT(2A)R and the SERT in rat forebrain. The forebrain regions affected are those which, in humans, are concerned with the control of mood, mental state, cognition and emotion. Here we have used the mixed estradiol agonist/antagonist, tamoxifen, to determine whether this action of estradiol is mediated by cytoplasmic estradiol receptors. Acute treatment ( approximately 32 h) of ovariectomized rats with estradiol benzoate (EB) increased significantly the amount of 5-HT(2A)R mRNA and SERT mRNA in the DRN and the densities of 5-HT(2A)R and SERT binding sites in the forebrain. These effects of EB were completely blocked by tamoxifen. Treatment with tamoxifen alone had no effect on either gene expression or the density of binding sites. Together, these data show that tamoxifen acts as a pure estradiol antagonist with respect to serotonergic mechanisms in brain. Detailed analysis of the effects of estradiol and tamoxifen on the DRN showed that SERT gene expression is constitutive only in the posterior DRN; in the anterior DRN, SERT gene expression appears to depend upon estrogen induction which is blocked by tamoxifen. Our findings strongly suggest that estradiol receptors are involved in mediating estradiol action on central serotonergic mechanisms and are relevant for our understanding of the effects of antiestrogens as well as estradiol on mood, mental state and cognition.

Nuclear and cytoplasmic maturation of mouse oocytes after treatment with synthetic meiosis-activating sterol in vitro.

Synthetically produced meiosis-activating sterol, a sterol originally derived from follicular fluid (FF-MAS), induces meiotic maturation of mouse oocytes in vitro. We therefore compared FF-MAS-induced maturation of naked mouse oocytes arrested in prophase I by either hypoxanthine (Hx) or forskolin (Fo) with spontaneous maturation of naked oocytes. FF-MAS-treated oocytes overcame the meiotic block by Hx or Fo, although germinal vesicle breakdown was delayed by 11 h and 7 h, respectively. We also investigated the influence of FF-MAS on chromosome, microtubule, and ultrastructural dynamics in Hx-cultured oocytes by immunocytochemistry and electron microscopy. Similarly to spontaneously matured oocytes, chromosomes became aligned, a barrel-shaped spindle formed, and overall organelle distribution was normal in FF-MAS-matured oocytes. The number of small cytoplasmic asters was elevated in FF-MAS-treated oocytes. Although the number of cortical granules (CGs) was similar to that in spontaneously matured oocytes, the overall distance between CGs and oolemma was increased in the FF-MAS group. These observations suggest that the initiation of meiotic maturation in FF-MAS-treated oocytes in the presence of high cAMP levels leads to a delayed but otherwise normal nuclear maturation. FF-MAS appears to improve oocyte quality by supporting microtubule assembly and by delaying CG release, which is known to contribute to reduced fertilization.

Meiosis-activating sterol promotes resumption of meiosis in mouse oocytes cultured in vitro in contrast to related oxysterols.

The sterol 4,4-dimethyl-5alpha-cholesta-8,14,24-trien-3beta-ol (FF-MAS [follicular-fluid meiosis-activating sterol]) from human follicular fluid has recently been identified as a compound that induces the resumption of meiosis. FF-MAS and various oxysterols have been reported to transactivate the orphan receptor LXRalpha. The objective was to determine the biological activity of synthetic FF-MAS on the resumption of meiosis and final maturation of mouse oocytes in vitro. In order to evaluate whether LXRalpha might mediate FF-MAS action on the oocyte, we compared the capability of various compounds to activate LXRalpha-dependent transcription and to induce resumption of meiosis in the oocyte assay. Ovaries were isolated from immature mice primed with FSH 48 h before collection. Naked oocytes (NkO) and cumulus enclosed oocytes (CEO) were isolated from follicles. The oocytes were cultured in two groups, NkO and CEO, respectively, in media containing either 3 mM hypoxanthine, 5 microM IBMX, or 0.100 mM dbcAMP to maintain the oocytes in the germinal vesicle stage. The resumption of meiosis was assessed by the frequency of germinal vesicle breakdown (GVBD) after 24 h of in vitro culture. FF-MAS overcame the meiotic inhibition by hypoxanthine in both the NkO group and CEO group in a dose-dependent manner within the concentration range 0.07-7 microM. FF-MAS displayed similar potency in all inhibitory agents used. Also, FF-MAS significantly increased the formation of polar bodies in both the CEO and NkO group. The oxysterols 22(R)-hydroxycholesterol (a potent ligand for the LXRalpha receptor), 16-hydroxycholesterol, 25-hydroxycholesterol, and 27-hydroxycholesterol, as well as cholesterol, were tested without any significant effect on maturation compared to that of controls. Oxysterols and FF-MAS were observed to activate LXRalpha. In conclusion, the results reported here clearly demonstrate that synthetic FF-MAS exclusively is capable of mediating resumption of meiosis in vitro in both NkO and CEO irrespective of the inhibitory substance used. In contrast, the oxysterols and cholesterol had no significant biological activity on this oocyte function, and consequently we found no correlation between LXRalpha activation and meiosis stimulation.

Proliferation and apoptosis in follicles of the marmoset monkey (Callithrix jacchus) ovary.

Proliferation and apoptosis were studied in ovarian follicles of immature and pubertal marmosets and in mature marmosets during the follicular, periovulatory and luteal phases. Proliferation was evaluated using a Ki 67 antibody and apoptosis was assessed by in situ detection of DNA fragmentation. In the immature animals only small follicles were present, and the expression of Ki 67 was restricted to the granulosa cells of follicles localised near the medulla. There was no evidence of DNA fragmentation. In pubertal and adult animals Ki 67 expression was found in the granulosa cells of some but not all primordial and primary follicles. In the secondary and tertiary follicles immunoreactivity was localized in theca cells and granulosa cells. In atretic follicles (morphologically classified) the number of Ki 67 positive granulosa cells varied. In corpora lutea as well as in corpora lutea accessoria, staining was seen in the nuclei of some luteal cells. During all phases of the cycle, follicles from the secondary stage onwards were proliferating, whereas granulosa cells of primary follicles were only stained during the follicular phase. During all phases of the ovarian cycle apoptosis was restricted to the granulosa cells of tertiary follicles. With regard to proliferation and apoptosis, follicles exhibiting morphological signs of atresia can be classified as follows: (1) granulosa cells showing strong Ki 67 expression; (2) granulosa cells with reduced expression of Ki 67; (3) granulosa cells devoid of Ki 67 immunoreactivity and of apoptotic signs; (4) granulosa cells heavily stained for DNA fragmentation and not stained for Ki 67; (5) granulosa cells close to the antrum showing DNA fragmentation but luteinizing Ki 67 positive granulosa cells close to the basement membrane. In summary, it was shown that atresia of tertiary follicles is characterised by three consecutive stages: morphological alterations, cessation of proliferation and finally apoptosis in tertiary follicles. Thus, our results indicate that early atresia as evidenced by the morphological signs is not necessarily related to DNA fragmentation, since apoptosis is exclusively found in the granulosa cells of advanced atretic tertiary follicles.

Effects of a pure antiestrogen and progesterone on estrogen-mediated alterations of blood flow and progesterone receptor expression in the aorta of ovariectomized rabbits.

There is ample evidence from epidemiological studies that estrogen-replacement therapy protects postmenopausal women against cardiovascular disease. One explanation for this beneficial effect could be the improvement of blood flow under estrogen therapy. By using ultrasound and Doppler color flow mapping we demonstrated in the aorta of ovariectomized rabbits a significant dose-dependent increase in blood flow after treatment with 17beta-estradiol. An increase in blood flow was already observed within 1 h of estradiol treatment and lasted until the end of a 14-day treatment phase. Progesterone did not attenuate the effects of 17beta-estradiol on aortic blood flow. The pure estrogen receptor antagonist ZM 182780, however, dose-dependently reversed the effect of 17beta-estradiol on blood flow after the 14-day treatment phase, but was not able to antagonize the rapid 17beta-estradiol effect on blood flow after 1 h. After killing the animals mRNA and protein expression of the progesterone receptor (PR), a known estrogen-responsive gene in classic target organs, were examined. Analogous to the blood flow results the PR mRNA level increased dose-dependently after 17beta-estradiol treatment, whereas ZM 182780 was able to reverse this effect. Immunohistochemical localization of PR in the aortic wall revealed an increase in immunoreactivity in fibroblasts of the adventitia after 17beta-estradiol treatment. ZM 182780, and to a lesser degree progesterone, reversed the 17beta-estradiol-induced increase in PR immunoreactivity. PR immunoreactivity was further detected in endothelial and smooth muscle cells, but the various hormonal treatments had no discernible effect on the PR mRNA level in these cellular compartments. Our findings in the aorta of OVX rabbits suggest that (a) 17beta-estradiol exhibits a rapid effect on arterial tone, (b) the pure estrogen receptor antagonist ZM 182780 inhibits the 17beta-estradiol effect on blood flow and PR mRNA and (c) progesterone does not attenuate the beneficial effect of estrogens on arterial tone.

Sexual dimorphism of steroid hormone receptor messenger ribonucleic acid expression and hormonal regulation in rat vascular tissue.

Evidence has accumulated suggesting that steroid hormones have a direct effect on the vascular system. Of special interest is the protective effect of estrogens against cardiovascular diseases. One of the aims of the present study was to investigate the messenger RNA (mRNA) expression of the five classic steroid hormone receptors in the great vessels of the rat (aorta, vena cava, and vena portae) to provide a basis to analyze direct steroid effects on vascular tissue. By applying reverse transcription-PCR we compared the expressions of the steroid hormone receptor mRNAs in the respective vessels of male and female rats. Sex differences in the mRNA levels of the mineralocorticoid (MR), estrogen (ER), and progesterone (PR) receptors were found in venous vessels, but not in the aorta. Focussing on the vena cava in the female rat, we investigated whether the ER is active in the vasculature by analyzing regulation of the PR gene. This gene is known to be regulated by estrogens in classic target organs. PR mRNA expression in venous vessels of female rats decreased after ovariectomy. This effect was reversed by chronic sc treatment with estradiol (E2; 1 microgram/animal day). Progester-one (10 mg/animal day, sc) partly inhibited the effect of E2. Besides E2, the partial agonist tamoxifen stimulated PR mRNA expression in ovariectomized rats, whereas the pure antiestrogen ZM 182780 remained inactive in this experiment. Both E2 and tamoxifen caused an autologous down-regulation of ER mRNA. In conclusion, our study demonstrates for the first time that not only the aorta, but also the venous vessels, represented by the vena cava and the portal vein of the rat, are targets for steroid hormones. The ER in vascular tissue is functionally active and mediates direct modulatory effects of estrogens on gene expression in vascular cells.

Identification of estrogen regulated genes in Fe33 rat hepatoma cells by differential display polymerase chain reaction and their hormonal regulation in rat liver and uterus.

We applied the differential display RT-PCR (ddRT-PCR) technology to identify estrogen-regulated hepatic genes in the estrogen receptor expressing rat hepatoma cell line Fe33. Three genes of known sequences were detected by the ddRT-PCR approach: IGF binding protein-1 (IGFBP-1), vitamin D-dependent calcium-binding protein (CaBP9k) and major acute phase protein (MAP). Effects of ethinyl estradiol on the mRNA levels of these genes were confirmed by "Northern-blot" analysis. If given in combination with dexamethasone and glucagon, ethinyl estradiol caused 40-, 15- and 11-fold increases in the mRNA steady state level of IGFBP-1, CaBP9k and MAP, respectively, in Fe33 cells 24 h after addition of hormone. Besides ethinyl estradiol, the partial estrogen agonist OH-tamoxifen caused dose dependent effects on expression of MAP and IGFBP-1. Estrogen regulation of the respective genes and the modulatory effects of progesterone (10 mg/animal/day) were studied in ovariectomized rats treated subcutaneously for 14 days with 1 microgram/animal/day estradiol. "Northern-blot" analysis of liver RNA revealed a 6-fold stimulation of IGFBP-1 mRNA levels in estradiol-treated compared to vehicle-treated rats and a weak but detectable increase of MAP mRNA steady state level (1.6-fold) upon estradiol administration. No effect of estradiol treatment could be monitored for CaBP9k in rat liver. Modulatory effects of progesterone on estradiol-stimulated expression in the liver could be monitored for IGFBP-1 only. In an extension of our investigation on the expression of the three genes in rat liver, we determined their expression and hormonal regulation in the uterus of the same animals. In the uterus, estradiol caused an increase in CaBP9k mRNA. In contrast, IGFBP-1 mRNA levels increased dramatically upon progesterone administration, whereas no effect of estradiol treatment could be detected. MAP mRNA levels increased only after coadministration of estradiol and progesterone. In conclusion, the ddRT-PCR proved to be a powerful method to identify estrogen-regulated genes. The study on the hormonal regulation of three genes stimulated by estrogen in Fe33 cells revealed similarities and differences in their regulation in vivo and in vitro.

Effects of the progesterone antagonists onapristone (ZK 98 299) and ZK 136 799 on surgically induced endometriosis in intact rats.

The effects of the progesterone antagonists (antiprogestins) onapristone (ZK 98 299) and ZK 136 799 on surgically induced endometriosis were studied in intact female rats. Endometriosis was induced by transplanting homologous endometrium to the parietal peritoneum of the abdominal wall (location A) and to the mesentery of the small intestine (location B). The animals were treated daily for 4 weeks at doses of 0.4 and 2.0 mg onapristone or ZK 136 799. The growth of the endometriosis-like foci was measured with a calliper during both pre- and post-treatment laparotomy. Both antiprogestins exerted inhibitory effects on the growth of the endometriosis-like foci in terms of complete remission. A 40 and 50% remission of endometriosis was observed at each location after the administration of 2.0 mg onapristone, whereas 50 and 63% (location A) and 50 and 75% (location B) remissions were found after the administration of 0.4 and 2.0 mg of ZK 136 799 respectively. ZK 136 799 was also more potent than onapristone in growth inhibition (85 versus 48% for location B) in animals with persistent endometriosis. Growth inhibition of the endometriosis-like foci was confirmed by histology and immunohistochemical staining of the proliferating cell nuclear antigen. The antiprogestins caused a reduction in glandular and luminal epithelial cells in the ectopic endometrium. Both antiprogestins tended to cause a decrease in uterine weight. Unlike the inhibitory effects in the ectopic endometrium, both onapristone and ZK 136 799 exhibited some stimulatory effects on the epithelial cells within the eutopic endometrium. Serum 17 beta-oestradiol concentrations did not vary significantly among the different treatment groups. No antiglucocorticoid effect of the antiprogestins was observed at either dose. This study indicates that the antiprogestins onapristone and ZK 136 799 exhibit antiproliferative effects in the ectopic but not the eutopic endometrium via mechanisms which remain to be established. The better efficacy of ZK 136 799 is more likely caused by its higher antiprogestagenic activity than its partial androgenic activity. These findings may be a further indication of the future potential of antiprogestins such as onapristone and ZK 136 799 in the treatment of endometriosis.

Physiological dilation of uteroplacental arteries in the guinea pig depends on nitric oxide synthase activity of extravillous trophoblast.

The trophoblast invasion of uteroplacental arteries in the guinea pig has been studied by means of electron microscopy and immunohistochemisty. To identify trophoblast cells, smooth muscle cells, and endothelial cells, antibodies against cytokeratins, smooth muscle myosin, desmin, and vimentin were employed. Furthermore, the immunohistochemical expression patterns of nitric oxide synthase isoforms (eNOS, mNOS and bNOS) were studied and were compared with the enzyme histochemical staining for NADPH-diaphorase. Dilation of uteroplacental arteries begins prior to day 30, when trophoblast cells that coexpress endothelial and macrophage nitric oxide synthase can be found in the vicinity of the vessels and replace the surrounding peritoneal mesothelium. Trophoblast invasion of the arterial walls and the subsequent wall destruction are only secondary effects. Starting around day 50, the final steps of pregnancy-dependent vessel modifications involve intraarterial trophoblast adhesion to the endothelium and subsequent replacement of the endothelium by the trophoblast cells. These may centrifugally invade the vessel media eventually forming intraluminal plugs. These findings led us to the conclusion that in the guinea pig pregnancy-induced physiological dilation of the uteroplacental arteries is due to the effect of nitric oxide rather than being caused by trophoblast-induced media destruction.

Comparative progestational activity of norgestimate, levonorgestrel-oxime and levonorgestrel in the rat and binding of these compounds to the progesterone receptor.

The progestational activity of norgestimate (NORG), levonorgestrel-oxime (LNG-oxime) and levonorgestrel (LNG) were compared in a pregnancy maintenance study in rats. The compounds were administered subcutaneously to pregnant rats at several doses, blood samples were collected repeatedly, and the concentration of LNG was measured in these samples. It could be demonstrated that following the administration of NORG and LNG-oxime, LNG was a major metabolite present in the serum. The pharmacological response in rats treated with NORG and LNG-oxime could be related to the systemic exposure of these animals to metabolically derived LNG. Thus, both NORG and LNG-oxime can be regarded as pro-drugs of LNG, the latter being almost exclusively responsible for the pharmacological activity of both pro-drugs. This notion was further supported by studies on the comparative binding affinity of these compounds to rabbit and human progesterone receptor (PR). LNG exhibited the highest binding affinity of the compounds studied. Relative binding affinity (RBA) values of LNG using progesterone as reference (100%) were found to be 125% for rabbit PR (rPR), 143% for human uterine PR (hPR) and 125% for recombinant hPR, respectively. In contrast to LNG, NORG exhibited only a low affinity to the PR, which is documented by RBA values of 1.2% for rPR, 3.2% for uterine hPR and 9% for recombinant hPR. The corresponding values of LNG-oxime were 30% (rPR), 20% (uterine hPR) and 18% (recombinant hPR), respectively. Thus, the combined experimental evidence of the present study does not support the view of NORG being a progestogen on its own as has been suggested by others.

Cervical ripening with the cytokines interleukin 8, interleukin 1 beta and tumour necrosis factor alpha in guinea-pigs.

It has been suggested that the collagenolytic enzymes released from white blood cells which infiltrate the pregnant human uterine cervix at term are responsible for connective tissue changes which take place during the ripening process. Similarly, an infiltration of inflammatory cells occurs in pregnant guinea-pigs either spontaneously at term or at preterm after treatment with the antiprogestin onapristone. The objective of this study was to evaluate the effects of the inflammatory cytokines interleukin 8 (IL-8), interleukin 1 beta (IL-1 beta), tumour necrosis factor alpha (TNF-alpha) and a combination of IL-1 beta and TNF-alpha on cervical ripening in guinea-pigs during advanced pregnancy. The cytokines were applied locally (intracervically) in a gel for 2 days and the effects were assessed on the third day by both extensibility measurements and morphological evaluation. IL-8 treatment on days 42 and 43 post coitum (p.c) and on days 48 and 49 p.c. (term: day 67 +/- 3 p.c.) significantly (P < 0.05) increased cervical extensibility at both stages of pregnancy. Although IL-1 beta treatment (days 42 and 43 p.c.) led to a slight increase in cervical extensibility, this effect was not statistically significant. An electron microscope study performed on days 48 and 49 p.c. revealed a pronounced cervical ripening accompanied by the dissolution of collagen fibres, stromal oedema and the infiltration of polymorphonuclear leukocytes in all cytokine-treated groups. The morphological effects of IL-8 and IL-1 beta were indistinguishable from those observed during normal cervical ripening at term.(ABSTRACT TRUNCATED AT 250 WORDS)