Christa Hegele-Hartung

Improvement of Endothelial Dysfunction by Selective Estrogen Receptor-α Stimulation in Ovariectomized SHR

Abstract—Both known estrogen receptors, ER&agr; and ER&bgr;, are expressed in blood vessels. To gain further insight into the role of ER&agr; in a functional setting, we investigated the effect of the novel highly selective ER&agr; agonist Cpd1471 on vascular reactivity in ovariectomized spontaneously hypertensive rats (SHR). After ovariectomy or sham operation, 12-week-old female SHR received either 17&bgr;-estradiol (E2, 2 &mgr;g/kg body wt per day), the selective ER&agr; agonist Cpd1471 (30 &mgr;g/kg body wt per day), or placebo. Acetylcholine-induced endothelium-dependent vasorelaxation was significantly blunted in aortas from ovariectomized rats (Rmax, 53%±3% versus sham, 79%±2%; P <0.001). Treatment with E2 or Cpd1471 significantly augmented acetylcholine-induced relaxation in ovariectomized rats (Rmax, 70%±2%; resp, 73%±2%). Endothelium-independent relaxation induced by sodium nitroprusside was not different among the four groups. The contractile response induced by the nitric oxide (NO) synthase inhibitor N&ohgr;-nitro-l-arginine, an index of basal NO formation, was significantly lower in ovariectomized rats compared with sham-operated animals (53±2% versus 77%±5%; P <0.01) and was normalized by both E2 (70%±2%) and Cpd1471 (70%±3%). Aortic endothelial NO synthase (eNOS) expression and phosphorylation of the vasodilator-stimulated phosphoprotein, an index of NO/cGMP-signaling, was reduced in ovariectomized SHR and normalized by E2 and Cpd1471. In SHR after ovariectomy, endothelium-dependent NO-mediated vasorelaxation and eNOS expression are attenuated. The novel selective ER&agr; agonist Cpd1471 prevented these pathophysiological changes to a similar extent as E2. Thus, the pharmacological principle of selective ER&agr; activation mediates positive vascular effects.

Both Estrogen Receptor Subtypes, α and β, Attenuate Cardiovascular Remodeling in Aldosterone Salt–Treated Rats

Experimental and population-based studies indicate that female gender and estrogens protect the cardiovascular system against aldosterone-induced injury. Understanding the function of estrogens in heart disease requires more precise information on the role of both estrogen receptor (ER) subtypes, ER&agr; and ER&bgr;. Therefore, we determined whether selective activation of ER&agr; or of ER&bgr; would confer redundant, specific, or opposing effects on cardiovascular remodeling in aldosterone salt–treated rats. The ER&agr; agonist 16&agr;-LE2, the ER&bgr; agonist 8&bgr;-VE2, and the nonselective estrogen receptor agonist 17&bgr;-estradiol lowered elevated blood pressure, cardiac mass, and cardiac myocyte cross-sectional areas, as well as increased perivascular collagen accumulation and vascular osteopontin expression in ovariectomized rats receiving chronic aldosterone infusion plus a high-salt diet for 8 weeks. Uterus atrophy was prevented by 16&agr;-LE2 and 17&bgr;-estradiol but not by 8&bgr;-VE2. Cardiac proteome analyses by 2D gel electrophoresis, mass spectrometry, and peptide sequencing identified specific subsets of proteins involved in cardiac contractility, energy metabolism, cellular stress response and extracellular matrix formation that were regulated in opposite directions by aldosterone salt treatment and by different estrogen receptor agonists. We conclude that activation of either ER&agr; or ER&bgr; protects the cardiovascular system against the detrimental effects of aldosterone salt treatment and confers redundant, as well as specific, effects on cardiac protein expression. Nonfeminizing ER&bgr; agonists such as 8&bgr;-VE2 have a therapeutic potential in the treatment of hypertensive heart disease.

Treatment of infertility

J Sùnksen*, DA Ohl, H Momose, FT Rocha, TEP Barros and F Biering-Sùrensen Department of Urology, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark, Department of Surgery, Section of Urology, University of Michigan, Ann Arbor, Michigan, USA; Department of Urology, Hoshigaoka Koseinenkin Hospital, Osaka, Japan; Spinal Injury Unit, University of SaÄo Paulo, SaÄo Paulo, Brazil; Centre for Spinal Cord Injured, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark

Distribution of estrogen and progesteron receptors in the uterus: an immunohistochemical study in the immature and adult pseudopregnant rabbit.

SummaryIn order to clarify the distribution and content of estrogen (ER) and progesteron receptors (PR) under changing hormonal influences within the various cell populations of the uterus (glandular and luminal endometrial epithelium, stroma, myometrium), immunohistochemical determinations using specific monoclonal antibodies were made. To correlate the immunohistochemical findings with peripheral hormone levels and specific tasks of the endometrium, 17β-estradiol and progesterone serum levels were measured and cell proliferation determined by use of BrdU-labelling-immunohistochemistry. At the subcellular level ER and PR were located exclusively in the cell nuclei of female rabbits, which were either immature and lacking any peripheral hormone levels or were pseudopregnant (d0–d8 p.hCG). In the immature rabbits a general faint ER and PR immunostaining was found. In addition to a general increase in ER and PR in all cell populations estrous rabbits (d0 p.hCG) showed a significant rise of ER in the epithelial cells and of PR in the myometrium. Within the epithelial cells and the myometrium the ER dropped heavily within a few days of pseudopregnancy. The PR, however, increased sharply during the first two days of pseudopregnancy and decreased gradually following d4 p.hCG. A close relationship was observed between the high PR content and the proliferation rate of the epithelial cells on d2 p.hCG. In spite of the more rapid decrease of ER compared with PR, the glandular epithelium retained positive immunostaining. In the stroma the ER and especially PR content did not change significantly during the course of pseudopregnancy suggesting that some of the well-known differentiation events in the luminal epithelium may be mediated by the stroma.

Immunocytochemical localization of uteroglobin in the rabbit endometrium

SummaryUteroglobin, the progesterone dependent pregnancy-characteristic endometrial protein in the rabbit, is found within the endometrial epithelium on the fourth and sixth day of pregnancy at the electron-microscopic level by use of the immunoperoxidase technique and a specific anti-uteroglobin serum from the sheep. As known from earlier studies, uteroglobin is the predominant protein synthesized of the endometrial secretion. In the present study, it is localized exclusively in the non-ciliated epithelial cells. A common route of secretory proteins within these cells is observed by uteroglobin labelling: rough endoplasmatic reticulum → Golgi complex → condensing vesicles → secretory products. Uteroglobin occurs in small vesicles on the trans-face of the Golgi complex, and in addition beneath the apical plasma membrane where it appears in membranebound vesicles, which apparently are extruded into the unterine lumen. Most of the uteroglobin is located in the luminal secretion. The distribution of intracellular uteroglobin is found only in cells of the basal endometrial gland, adjacent to the myometrium. The cytoplasm of uterine epithelial cells facing the cavum does not show uteroglobin reaction products.

Effects of visible light and room temperature on the ultrastructure of preimplantation rabbit embryos : a time course study

SummaryIn a time course study (4–20 h) rabbit early cleavage stages (day 1 p.c.) and compacted morulae (day 3 p.c.) were exposed to visible light or room temperature (23° C), respectively. An 8 h light exposure of day 1 embryos caused alterations in nuclear morphology (lobulated nuclei, loss of nucleolar diffentiation), an increased electron density of the cytoplasm, and cellular fragmentation leading to a considerable degeneration of blastomeres (central clustering of organelles, loss of cell surface differentiation) after a 20 h exposure. Room temperature exposure (compacted Day 3 morulae) led to decompaction and a cleavage delay after 8 h. After 10 h, arrested metaphases occurred in all examined morulae. Even after 20 h at 23° C, day 3 embryos were at the decompacted morula stage, and showed metaphase-arrested blastomeres. The general morphology of the blastomeres was unaffected at this temperature, except for vacuolated serand cis-side vesicles of the Golgi complex at 8, 12 and 20 h, respectively.

Differential Effects of 17β-Estradiol and of Synthetic Progestins on Aldosterone-Salt–Induced Kidney Disease

Elevated mineralocorticoid levels and female sex hormones have been shown to confer opposing effects on renal injury, but their combined effects are still unknown. Objective: Identify the function of estrogens and of different synthetic progestins on aldosterone salt–mediated renal disease. Methods: The role of 17β-estradiol, medroxyprogesterone acetate (MPA), and drospirenone during renal injury was studied in Wistar rats subjected to uni-nephrectomy plus aldosterone salt treatment. Results: Aldo-salt treatment of intact, ovariectomized, and estradiol-treated female rats resulted in remnant kidney hypertrophy without structural damage. Co-treatment with MPA, but not with drospirenone, increased kidney hypertrophy, fluid turnover, sodium retention, and potassium excretion. Medroxyprogesterone acetate also caused glomerular, vascular, tubular, and interstitial lesions that were accompanied by increased blood pressure and enhanced NADPH oxidase (p67phox) and sodium channel (α-ENaC) expression. Drospirenone, a progestin with anti-mineralocorticoid function, and spironolactone prevented kidney hypertrophy, hypertension, and sodium retention. Drospirenone and spironolactone also increased renal angiotensin II type 2 receptor expression and relieved aldosterone-induced suppression of serum angiotensin II levels. Conclusion: The choice of specific synthetic progestins has profound implications on the development of kidney injury and renal gene expression under conditions of elevated aldosterone serum levels and salt intake.

Ultrastructure of preimplantation rabbit embryos exposed to visible light and room temperature

SummaryEarly cleavage stage embryos (day 1 p.c.) and morulae (day 3 p.c.) of rabbits were exposed to visible (standard) lighting (1600 lx) and room (standard) temperature (23°C) during a 24 h in-vitro culture. Control embryos were cultured in darkness at 37°C. Development was assessed by light and electron microscopy as well as by the cytochemical demonstration of glycogen.In day 1 and day 3 embryos standard temperature induced swelling of the SER and Golgi complex vesicles. Major changes in day 1 embryos consisted of smallish microtubules-like crystalloids, and in day 3 embryos of unusually large SER vesicles. In both embryonic ages cleavage rate and development was more retarded by standard temperature than by standard lighting. Standard lighting, however, led to distinct signs of degeneration and cell death. The mode of cell damage seemed to be different in light exposed early cleavage stages and morulae: In day 1 embryos cytoplasmic degeneration was predominant while the majority of cells in day 3 embryos died by apoptosis. Despite clear indications of cell damage, cleavage rate was not notably impaired compared with non-exposed controls. Glycogen increased during development from cleavage stages to early blastocysts. The distribution was not changed either by exposure to standard temperature nor by standard lighting.The results demonstrate that day 1 embryos were clearly more susceptible to lighting whereas day 3 embryos were more affected by temperature. The mode of damage exerted by both the physical environmental factors was different. Reduction to standard temperature interfered mainly with the organization of the cytoskeleton and intracellular transport of organelles, while exposure to standard lighting led to cell degeneration and death.

Effect of in vitro culture on the dynamics of uteroglobin distribution in rabbit blastocysts.

SummaryThe purpose of this study was to investigate the localization and transport of uteroglobin in normal rabbit blastocysts (day 4-day 6 p.c.) and in those cultured for 6–48 h in vitro, using a specific radioimmunoassay and immunocytochemistry. The results of the radioimmunoassay showed that in day 4 p.c. blastocyst tissue (based on homogenate measurements) a significant decrease of the uteroglobin content started after only 6 h of culture in vitro. A significant concomitant rise of uteroglobin was observed in the culture medium after 12 h of in vitro culture. Using immunocytochemistry it was not possible to detect uteroglobin in any compartment of the non-cultured or in vitro cultured day 4 p.c. blastocysts. The efflux of uteroglobin down a concentration gradient was confirmed by the immunocytochemistry in non-cultured and in vitro cultured day 5 p.c. and day 6 p.c. blastocysts. Uteroglobin immunoreactions were mainly detected in non-cultured blastocysts (day 5 and 6 p.c.) in large vesicles of the trophoblast cells. In addition endocytotic vesicles at the inside of the apical membrane of trophoblast cells, some cell debris within the perivitelline space and the neozona were labelled. During in vitro culture of day 5 and 6 p.c. blastocysts, uteroglobin labelling in the coverings did not change. In non-cultured and cultured day 5 and 6 p.c. blastocysts neither the compartments of the embryoblast, the endoderm cells nor the blastocyst cavity showed any uteroglobin immunoreactions. After only 6 h of in vitro culture, uteroglobin immunoreactions were no longer found within the trophoblast cells. The reaction did not reappear during the course of in vitro culture up to 48 h, suggesting a complete lack of de novo synthesis of uteroglobin by blastocysts.

Ultrastructural and Autoradiographic study of Preimplantation rabbit embryos grown in conventional or uterine flushing-supplemented culture media

Rabbit morulae and blastocysts were cultured in conventional culture media [Ham’s F10 or BSM II supplemented with bovine serum albumin (BSA) or serum] or in Ham’s medium supplemented with synchronous or asynchronous uterine flushings, mostly for 2 days, and afterwards investigated by light and electron microscopy and by autoradiography. Ultrastructure and cell proliferation differed considerably between cultured embryos and noncultured controls. Cultured embryos displayed more dead cells. They were developmentally retarded (predominance of smooth endoplasmic reticulum rather than the age-specific rough endoplasmic reticulum, mitochondria still round to ovoid shaped) and showed nonspecific signs of cells damage (swollen mitochondria and Golgi complex vesicles, increased number of lysosomes). All these features were also present in embryos grown in uterine flushing-supplemented media, but were less pronounced. Cell damage and impaired cell proliferation had affected trophoblast cells more than embryoblast cells. Endoderm could be differentiated only if culture had been started with blastocysts—not with morulae—and seems to require uterine secretions. No significant ultrastructural differences were observed between embryos cultured in synchronous or in asynchronous uterine flushings. Present results indicate that cultured preimplantation rabbit embryos deviate clearly from those grown in vivo and maintain, for some time, a better cellular structure—and probably function —in the presence of uterine flushings than in conventional culture media. Specific abnormal morphologic features related to a particular medium could not be identified.