Adiponectin induces periovulatory changes in ovarian follicular cells.

Adiponectin, the most abundantly synthesized protein in adipose tissue, has plieotropic effects on liver, muscle, endothelium, placenta, and other tissues. We examined direct effects of recombinant porcine adiponectin on porcine ovarian granulosa cells in vitro. We demonstrate that adiponectin, at physiologically relevant levels (10-25 microg/ml), provokes expression of genes associated with periovulatory remodeling of the ovarian follicle over a time frame of 6-24 h. These include cyclooxygenase-2, prostaglandin E synthase, and vascular endothelial growth factor. Adiponectin modulates steroid synthetic protein gene expression, increasing steroidogenic acute regulatory protein transcript abundance and reducing cytochrome P450aromatase. Adiponectin has antidiabetic properties and sensitizes tissues to insulin. We show that it interacts with both LH and insulin in inducing expression of cyclooxygenase-2 transcripts in granulosa cells. We determined that the MAPK pathway, via phosphorylation of ERK1/2, is involved in mediation of the adiponectin signal in ovarian granulosa cells, rather than protein kinase A or the classic adiponectin transducer, AMP-activated protein kinase. Adiponectin synthesis is reduced in obesity, and our findings suggest that this reduction plays a role in obesity-related ovarian dysfunction.

Transcriptional regulation of uterine vascular endothelial growth factor during early gestation in a carnivore model, Mustela vison.

Vascular endothelial growth factor (VEGF) is an essential angiogenic signaling element that acts through its two tyrosine kinase receptors, inducing both proliferation of endothelial cells and vascular permeability. Given the importance of vasculogenesis and angiogenesis to early pregnancy, it is of interest to understand the mechanisms regulating vascular development at this stage. We previously demonstrated that VEGF and receptors are up-regulated during embryo implantation in an unique animal model, the mink, a species displaying obligate embryonic diapause. Herein we examined the role of prostaglandin E2 (PGE(2)) as a regulator of VEGF during early pregnancy and established the mechanisms of this regulation. We demonstrate that activated embryos secrete PGE(2) and that expression of PGE synthase protein in the uterus is dependent upon direct contact with invading trophoblast cells during implantation. Using mink uterine stromal cells transfected with mink VEGF promoter driving the luciferase reporter gene, we show that PGE(2) induces promoter transactivation and that this response can be eliminated by blockade of protein kinase A. Treatment with antagonists to PGE(2) receptors EP2 and EP4 eliminated the PGE(2)-induced response in transfected cells. Deletional studies of the promoter revealed that a region of 99 bp upstream of the transcription start site is required for PGE(2)-induced transactivation. Mutation of an AP2/Sp1 cluster, found within the 99 bp, completely eliminated the PGE(2) response. Furthermore, chromatin immunoprecipitation assays confirmed binding of the AP2 and Sp1 transcription factors to the endogenous mink VEGF promoter in uterine cells. PGE(2) stimulated acetylation of histone H3 associated with the promoter region containing the AP2/Sp1 cluster. Taken together, these results demonstrate that PGE(2) plays an important role in regulating uterine and thus placental vascular development, acting through its receptors EP2 and EP4, provoking protein kinase A activation of AP2 and Sp1 as well as acetylation of histone H3 to transactivate the VEGF promoter.

Modulation of peroxisome proliferator-activated receptor delta and gamma transcripts in swine endometrial tissue during early gestation.

Recent evidence points to a role for peroxisome proliferator-activated receptors (PPARs) delta and gamma in embryo implantation and survival. In this study, we report the porcine PPARdelta complete coding sequence and mRNA abundance of PPARdelta, PPARgamma1 and gamma2, angiopoietin-like protein 4 (ANGPTL4) and adipocyte determination and differentiation-dependent factor 1 (ADD1) genes in the pregnant sow endometrium. Real-time PCR analysis was used to study the effect of parity (Yorkshire-Landrace multiparous (YL) and nulliparous (YLn)), site of endometrial tissue sampling (between and at embryo attachment sites) in crossbred DurocxYorkshire-Landrace (DYL) sows and stages of pregnancy (non-pregnant, day 15 and day 25 after mating) in Meishan-Landrace (ML) on mRNA levels. Parity effects were observed for PPARdelta, ANGPTL4, and ADD1, with higher mRNA levels in YL than YLn sows. In DYL sows, lower mRNA levels were present at attachment sites compared to between attachment sites for PPARdelta, PPARgamma1, and ANGPTL4. Finally, day 15 pregnant ML sows had lower PPARdelta mRNA levels compared to day 15 cycling ML sows. A significant increase of PPARgamma1 mRNA levels was found on day 25 pregnant ML and DYL sows relative to day 15 ML or DYL pregnant sows. PPARdelta and gamma immunostaining was detected in endometrial tissue of day 15 cycling sows, day 15 and 25 pregnant sows and epithelial cells of day 25 embryos. Collectively, our results suggest a role for PPARdelta, PPARgamma1, and ANGPTL4, but not PPARgamma2, during the peri-implantation period in pregnant sows.

Aberrant intracellular cholesterol transport disrupts pituitary and ovarian function.

Cholesterol is imported and processed to provide substrate for ovarian steroidogenesis. The Niemann Pick type C-1 gene codes for a glycoprotein that processes low-density lipoproteinimported cholesterol. Mutation of this gene causes marked impairment of export of low-density lipoprotein-derived cholesterol from endosomes, and consequent lysosomal accumulation of the sterol. The BALB/c npc(nih-/-) mouse line, bearing spontaneous mutation of the NPC-1 gene, provides a model for investigation of aberrant endosomal cholesterol transfer in the ovary. Female homozygote mutant mice are infertile, with underdeveloped ovarian follicles, reduced steroidogenesis, no ovulation, and no corpora lutea. Mutant ovaries transplanted under wild-type kidney capsules display both ovulation and formation of corpora lutea. Gonadotropin treatment induces ovulation and restores expression of steroidogenic proteins. Pituitary glands of mutants are hypoplastic, and prolactin expression is dramatically reduced compared with wild-type mice. Both long and short splice variants of the dopamine-D2 receptors are overexpressed in the pituitary of BALB/c npc(nih-/-) mice. Chronic treatment of mutant mice with 17beta-estradiol restores pituitary volume, prolactin expression, and folliculogenetic capability. We conclude that inactivating mutation of Niemann Pick C-1 perturbs the hypothalamic-pituitary-ovarian feedback loop. Reduced estrogens attenuate prolactin expression and alter gonadotropin secretion patterns and interfere with normal ovarian follicular development and ovulation.

Expression of vascular endothelial growth factor isoforms and receptors Flt-1 and KDR during the peri-implantation period in the mink, Mustela vison.

Expression of vascular endothelial growth factor (VEGF) isoforms and its receptors, Flt-1 and KDR, was investigated during the period of peri-implantation in mink, a species that displays obligate embryonic diapause. Uterine samples were collected during diapause, embryo activation, and implantation from pseudopregnant and anestrous animals and analyzed by semiquantitative reverse transcription polymerase chain reaction and immunohistochemistry. The abundance of mRNA of VEGF isoforms 120, 164, and 188 was highest during late embryo activation and at implantation. VEGF protein was localized to the glandular epithelium at all stages of peri-implantation, whereas the luminal epithelium lacked VEGF reactivity during diapause. Endometrial stroma and luminal and glandular epithelia were positive for VEGF in implanted uteri. The invasive trophoblast cells of the implanting embryo were intensively stained. High levels of VEGF mRNA in pseudopregnant uteri indicates that VEGF upregulation leading to implantation is dependent upon maternal rather than embryonic factors. The abundance of the two receptors, KDR and Flt-1, increased in the uterus during implantation. Low levels of the receptors in pseudopregnant uteri compared with those containing activated or implanted embryos indicates that the embryo regulates receptor expression. These results demonstrate VEGF and VEGF receptor expression during early gestation in mink and suggest that maternal and embryonic input regulates different aspects of the angiogenic process.

Estratégias de reperfusão precoce no infarto agudo do miocárdio / Early reperfusion strategies in acute myocardial infarction

PURPOSE: To describe the use of myocardial reperfusion strategies (percutaneous transluminal coronary angioplasty-PTCA and intravenous trombolysis) whenever it is possible to use emergency cinecoronariography in acute myocardial infarction (AMI). METHODS: The records of patients admitted with initial diagnosis of AMI, until six hours after the beginning of symptoms, were reviewed retrospectively, between March/92 and December/93. RESULTS: One hundred forty-three patients were admitted with suspected AMI. Eighty-one (57) presented definitive criteria for this diagnosis. Fifty-two patients (64) were admitted in the first six hours from the beginning of symptoms. Thirty-eight (73) were male and the mean age was 59 +/- 12 years. Cinecoronariography was indicated immediately in 36 of 52 (69) patients, with purpose to use PTCA. That was done in 30 (58). Two (4) patients were submitted to emergency surgical myocardial revascularization. Intravenous thrombolysis was used in 11 (21) patients. At admission, the conservative treatment was chosen for five out of nine left patients, based on clinical grounds. CONCLUSION: The availability of emergency cinecoronariography made an early reperfusion strategy possible in 83 of patients admitted with AMI in the first six hours after the beginning of symptoms

Objetivo - Descrever a utilização de estratégias de reperfusão miocárdica (angioplastia transluminal coronária primária - ATCP e a trombólise intravenosa) e o emprego da cinecoronariografia de emergência no infarto agudo do miocárdio (IAM). Métodos - Foram revistos, retrospectivamente, entre março/92 e dezembro/93, os prontuários dos pacientes admitidos com diagnóstico de IAM, com até 6h após o início dos sintomas. Resultados - Foram admitidos com suspeita de IAM, 143 pacientes dos quais 81 (57%) com critérios definitivos para este diagnóstico, sendo que 52 (64%) admitidos durante as 6 primeiras horas dos sintomas. Eram do sexo masculino 38 (73%), com média de idade de 59±12 anos. A cinecoronariografia foi indicada de forma imediata em 36 (69%) dos 52 pacientes, visando ATCP que foi realizada em 30 (58%). Dois (4%) pacientes foram encaminhados para cirurgia de revascularização miocárdica de emergência. A trombólise intravenosa foi utilizada em 11 (21%) pacientes. Na admissão optou-se pelo tratamento conservador em 5 dos 9 pacientes restantes, baseado no julgumento clínico. Conclusão - A disponibilidade da cinecoronariografia de emergência possibilitou a utilização de estratégias de reperfusão precoce em 83% dos pacientes admitidos com IAM durante as primeiras 6h