No abstract

No abstract

Placentation requires the production of numerous growth factors, hormones and transcription factors. Many of them, like the adipose tissue-derived leptin or adiponectin, have been identified in the placenta and their role has been established in the proliferation and subsequent development of the placenta. Apelin is another adipokine known for proliferative effects in different cell types. PcR, immunoblotting and immunocytochemistry were used to study mRNA and protein expression of apelin and its receptor (APJ) in syncytiotrophoblast (BeWo) and cytotrophoblast (JEG-3) cells as well in immunohistochemistry in human normal placenta slides. The effect of apelin on cell proliferation study was investigated by alamarBlue ® and cell counting Kit-8 assays, the cell cycle by the flow cytometry method and the protein expression of cyclins and phosphorylation level of extracellular signal-regulated kinases (ERK)1/2, phosphatidylinositol 3'-kinase/protein kinase B (Akt), signal transducer and activator of transcription 3 (Stat3) and 5'-monophosphate-activated protein kinase (AMPKα) were studied by western blotting. Apelin was increased in JEG-3 compared with in BeWo cells, while APJ was the same in both placenta cell lines. Immunocytochemical analyses revealed high cytoplasmic and/or membrane apelin localisation in JEG-3, while BeWo cells exhibited markedly weaker apelin signal in the cytoplasm. Apelin increased cell proliferation as well as the percentage of cells in the G2/M phase of the cell cycle, cyclin proteins and the expression of all kinases mentioned above. In conclusion, apelin by promotion of trophoblast cell proliferation by APJ and ERK1/2, Stat3 and AMPKα signalling could be a new important adipokine in the regulation of early placental development.

Previously, we demonstrated the expression of resistin in the porcine ovary, the regulation of its expression and its direct effect on ovarian steroidogenesis. The objective of this study was to examine the effect of resistin on cell proliferation and apoptosis in a co-culture model of porcine granulosa and theca cells. First, we analysed the effect of resistin at 1 and 10 ng/ml alone or in combination with FSHand IGF1 on ovarian cell proliferation with an alamarBlue assay and protein expression of cyclins A and B using western blot. Next, the mRNA and protein expression of selected pro-apoptotic and pro-survival regulators of cell apoptosis, caspase-9, -8 and -3 activity and DNA fragmentation using real time PCR, western blot, fluorescent assay and an ELISA kit, respectively, were analysed after resistin treatment. Furthermore, we determined the effect of resistin on the protein expression of ERK1/2, Stat and Akt kinase. Using specific inhibitors of these kinases, we also checked caspase-3 activity and protein expression. We found that resistin, at both doses, has no effect on cell proliferation. The results showed that resistin decreased pro-apoptotic genes, which was confirmed on protein expression of selected factors. We demonstrate an inhibitory effect of resistin on caspase activity and DNA fragmentation. Finally, resistin stimulated phosphorylation of the ERK1/2, Stat and Akt and kinases inhibitors reversed resistin action on caspase-3 activity and protein expression to control. All of these results showed that resistin has an inhibitory effect on porcine ovarian cell apoptosis by activation of the MAPK/ERK, JAK/Stat and Akt/PI3 kinase signalling pathways. Reproduction (2015) 150 343-355

Monocultures of different placental cells are used for many physiological and toxicological studies; however, they are not a true reflection of the interaction between placenta and fetus. To develop the most appropriate model to study endocrine and metabolic properties of fetoplacental unit we used three co-culture models of placental cells nonfusogenic JEG-3, unsyncytialised BeWo (BeWo) and syncytialised BeWo (syncBeWo) cultured with adrenal (H295R) cells. As an end point of endocrine properties we investigated steroids receptors expression and steroid secretion, while as metabolic properties AhR, CYP1A1and COMT expression. Progesterone (P4), estradiol (E2) and human chorionic gonadotropin (hCG) secretion (ELISA) and 3βHSD, CYP19, estrogen (ERα/β), progesterone (PR) and aryl hydrocarbon (AhR) receptors, CYP1A1 and COMT protein expression (Western blot) were evaluated. Comparing three co-culture models we observed: (1) there were no differences between JEG-3 and BeWo in the PR expression, however it was higher in BeWo compared to syncBeWo; (2) there were no differences in ERα protein expression in all models, while profile of ERβ expression was the highest in syncBeWo; (3) high P4 secretion in JEG-3 and BeWo while low in syncBeWo; (4) high E2 levels in JEG-3 and syncBeWo, while low E2 secretion in BeWo; (5) the highest hCG secretion in the JEG-3 and syncBeWo than in BeWo (6) the highest AhR, CYP1A1 and COMT expression in syncBeWo. Based on the results showing higher hCG secretion in the JEG-3 than in BeWo, representing villous and extravillous phenotype we suggest that JEG-3 model could be used to study fetoplacental steroidogenesis at the 1st, while BeWo model at the 3rd. Results showing comparable profiles of AhR, CYP1A1 and COMT expression in JEG-3 and BeWo models and the significantly higher expression in synBeWo points to synBeWo as a good model for study the metabolic properties.Electronic supplementary materialThe online version of this article (doi:10.1007/s10616-017-0142-z) contains supplementary material, which is available to authorized users.

Resistin, a hormone secreted by adipocytes, is thought to be important in reproduction. Our previous study demonstrated resistin expression in porcine ovarian follicles and its direct effect on steroidogenesis. The aim of the current study was to evaluate the effect of gonadotropins and the local ovarian factors, such as insulin-like growth factor type 1 (IGF1) and steroids (progesterone, testosterone, and 17 beta-estradiol), on the expression and secretion of resistin, as well as its steroidogenic action. Porcine ovarian follicles were exposed to follicle-stimulating hormone (FSH) and luteinizing hormone (LH) at 50-150 ng/ml, IGF1 (10-100 ng/ml), and steroids at 10(-8) to 10(-6) M for 24 h. Then, mRNA, protein expression, and medium concentration of resistin were determined using real-time PCR, Western blot analysis, and ELISA, respectively. In the subsequent experiments, ovarian follicles were exposed to resistin and/or FSH, LH, IGF1, and steroids, and ovarian steroidogenesis was analyzed. Additionally, we examined the direct effect of resistin on the protein expression of receptors for gonadotropins and investigated local factors. The results showed that gonadotropins and steroids have stimulatory effects but that IGF1 has an inhibitory effect on resistin expression and secretion. Resistin decreased gonadotropins and local hormone-induced steroid secretion and inhibited 3beta-hydroxysteroid dehydrogenase, 17beta-hydroxysteroid dehydrogenase, and cytochrome P450 aromatase protein expression. Additionally, we demonstrated that resistin increased the expression of receptors for progesterone and testosterone. These findings all show that the expression and function of resistin are regulated by gonadotropins and local factors produced by ovarian follicles.

No abstract

In the present study, using real-time polymerase chain reaction and immunoblotting methods, we quantified the expression of peroxisome proliferator-activated receptor (PPAR) γ, PPARα and PPARβ in different sized ovarian follicles (small (SF), medium (MF) and large (LF) follicles) in prepubertal and adult pigs. In prepubertal pigs, PPARγ and PPARα expression was highest in LF; however, PPARβ expression did not differ among SF, MF and LF. In mature pigs, only protein expression of PPARγ and PPARα increased during ovarian follicle development. Following identification of very high levels of PPARγ expression in LF in prepubertal and adult pigs, using in vitro culture of ovarian follicles, we determined the effect of resistin at 0.1, 1 and 10ngmL(-1) on PPARγ mRNA and protein expression and the effect of rosiglitazone at 25 and 50µM (a PPARγ agonist) on resistin mRNA and protein expression. Resistin increased PPARγ expression in ovarian follicles in both prepubertal and adult pigs, whereas rosiglitazone had an inhibitory effect on resistin expression. The role of PPARγ in regulating the effects of resistin on ovarian steroidogenesis was investigated using GW9662 (a PPARγ antagonist at dose of 1μM). In these studies, GW9662 reversed the effect of resistin on steroid hormone secretion. The data suggest that there is local cooperation between resistin and PPARγ expression in the porcine ovary. Resistin significantly increased the expression of PPARγ, whereas PPARγ decreased resistin expression; thus, PPARγ is a new key regulator of resistin expression and function.