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Expression and role of resistin on steroid secretion in the porcine corpus luteum. Kurowska P et al. (2021) Resistin plays an important role in adipogenesis, obesity, insulin resistance and reproduction. Previous studies showed resistin action on ovarian follicular cells; however, whether resistin regulates steroid secretion in luteal cells is still unknown. Our aim was first to determine the expression of resistin and its potential receptors (tyrosine kinase-like orphan receptor 1 ROR1] and Toll-like receptor 4 [TLR4]) in the porcine corpus luteum (CL), regulation of its expression, effect on kinases phosphorylation and luteal steroidogenesis. Our results showed that the expression of resistin and its receptors was dependent on the luteal phase and this was at the mRNA level higher in the late compared with the early and middle luteal phase. At the opposite, resistin protein expression was higher in the middle and late compared with the early luteal phase, while ROR1 and TLR4 expression was highest in the early luteal phase. Additionally, we observed cytoplasmic localisation of resistin, ROR1 and TLR4 in small and large luteal cells. We found that luteinising hormone, progesterone (P4), insulin and insulin-like growth factor 1 regulated the protein level of resistin, ROR1 and TLR4. Resistin decreased P4 and increased oestradiol (E2) secretion via changing in steroidogenic enzymes expression and via the activation of protein kinase A (PKA) and mitogen-activated protein kinase (MAP3/1), increased the expression of receptors LHCGR and ESR2 and decreased the expression of PGR. Moreover, resistin decreased PKA phosphorylation and enhanced MAP3/1 phosphorylation. Taken together, resistin could act directly on steroid synthesis and serve as an important factor in in vivo luteal cell function.////////////////// Effect of resistin on estradiol and progesterone secretion from human luteinized granulosa cells in culture. [Messini CI et al. (2019) Information on the role of resistin on steroidogenesis is limited to animal studies. The aim of this study was to investigate the effect of various doses of resistin on estradiol and progesterone secretion from human luteinized granulosa cells in culture. Granulosa cells were obtained from follicular fluid aspirated from 50 women undergoing in vitro fertilization (IVF) treatment. The cells were cultured for 48 h after a 24 h pre-incubation period. The effect of resistin at dosages 1, 10 and 100 ng/ml alone or in combinations with FSH (10 and 100 ng/ml) on steroidogenesis was investigated. Estradiol and progesterone were measured by radioimmunoassays in culture supernatants at 24 h and 48 h. FSH treatment increased both estradiol and progesterone secretion. Resistin suppressed basal estradiol (at 1 ng/ml) and progesterone secretion (at all concentrations tested). When resistin (all concentrations) was combined with FSH (100 ng/ml), it eliminated the stimulatory effect of FSH on the secretion of estradiol and progesterone. This study indicates an inhibitory effect of resistin on the secretion of estradiol and progesterone by human luteinized granulosa cells in vitro. It is likely that this adipokine locally affects ovarian function in women. Abbreviations: 3β-HSD: 3β-hydroxysteroid dehydrogenase; CAP1: cyclase-associated protein 1; DCN: decorin; FIZZ: Found in Inflammatory Zones; hCG: human chorionic gonadotropin; IGF1: insulin-like growth factor type 1; IVF: in vitro fertilization; PCOS: polycystic ovary syndrome; RIA: radioimmunoassay; ROR1: receptor tyrosine kinase-like orphan receptor-1; TLR4: Toll-like receptor 4.//////////////////
Resistin is a survival factor for porcine ovarian follicular cells. Rak A et al. (2015) Previously, we demonstrated expression of resistin in the porcine ovary, regulation of their expression and the 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 FSH- and 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. Moreover, we determined 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 demonstrated 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.//////////////////
Effects of resistin on ovarian folliculogenesis and steroidogenesis in the vespertilionid bat, Scotophilus heathi. Singh A 2014 et al.
The bat Scotophilus heathi exhibit prolonged anovulatory condition known as delayed ovulation coinciding with the period of extensive fat accumulation. The present study was undertaken to find out whether extensive accumulation of fat in S. heathi is responsible for suppression of ovarian activity by increasing production of adipokine resistin in the bat. This was achieved by (a) investigating variation in serum resistin level in relation to the changes in the body fat mass and (b) evaluating the effect of resistin treatment on ovarian activity with reference to steroid synthesis. An attempt was also made to determine whether resistin mediate its effects on ovary through signal transducer and activator of transcription 3 (STAT3) signaling mechanism. The results showed significant seasonal variation in serum resistin level with the peak level coinciding with the period of maximum fat accumulation, high circulating androgen level and period of anovulation. The treatment with resistin to the bat caused increase in androstenedione due to stimulatory effects on 3-hydroxysteroid dehydrogenase, but decrease in estradiol level due to inhibitory effect on aromatase. Resistin treatment increased androgen receptor protein together with increased insulin receptor but not through conventional luteinizing hormone receptor and steroidogenic acute regulatory protein mediated pathways. This study further showed that resistin treatment increases androstenedione synthesis and up-regulates insulin receptor in the ovary through STAT3 mediated pathways. These findings suggest that obese women through increased resistin synthesis may causes development of non-ovulatory antral follicles through insulin receptor signaling cascade.
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A Role for Resistin in the Ovary During the Estrous Cycle. Rak-Mardyla A 2014 et al.
In a previous study, we showed that resistin expression increased during ovarian follicle development in prepubertal pigs and had direct effects on steroidogenesis, suggesting an important role for resistin in the ovary during puberty. To determine its potential regulatory role in the ovary during the estrous cycle, using real-time PCR, immunoblotting, immunohistochemistry, and ELISA methods, we quantified the expression, immunolocalization and concentration of resistin in different sized ovarian follicles (small, 2-4?mm; medium, 4-6?mm; and large, 8-12?mm) in mature pigs. We then determined the effects of recombinant resistin (0.1, 1, and 10?ng/ml) on steroid hormone (progesterone-P4, androstendione-A4, testosterone-T, and estradiol-E2) secretion and steroidogenic enzyme (3?SD, CYP17A1, 17?SD, and CYP19A1) gene and protein expression in ovarian follicles. We found no differences in the resistin expression between all of the examined follicles. Immunostaining analysis also showed resistin expression in the cytoplasm of both granulosa and theca cells, where it was localized more abundantly in the granulosa cells compared to the theca cells. Recombinant resistin direct stimulated P4, A4, and T secretion via increased expression of 3?SD, CYP17A1, and 17?SD, suggesting an autocrine and/or paracrine regulatory role in the porcine ovary during the estrous cycle.
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Resistin decreases insulin-like growth factor I-induced steroid production and insulin-like growth factor I receptor signaling in human granulosa cells. Reverchon M et al. OBJECTIVE: To identify resistin in human ovarian follicles and investigate the effect and the molecular mechanisms associated with resistin on steroidogenesis in human granulosa cells (GCs). DESIGN: The effects of recombinant human resistin on the secretion of progesterone (P) and estradiol (E2) by cultured human GCs were investigated. SETTING: Academic institutions. PATIENT(S): Twenty infertile and healthy women undergoing IVF. INTERVENTION(S): Primary human GC cultures stimulated with recombinant human resistin (10 ng/mL). MAIN OUTCOME MEASURE(S): Determination of messenger RNA (mRNA) and protein expression of resistin in fresh human GCs by reverse transcriptase-polymerase chain reaction (RT-PCR), immunoblot and immunohistochemistry, respectively; measurement of P?and E2 levels in the conditioned media by radioimmunoassay; determination of cell proliferation by tritiated thymidine incorporation; and analysis of signaling pathways activation by immunoblot analysis. RESULT(S): Human GCs and theca cells express resistin. In primary human GCs, resistin decreases P and E2 secretion in response to insulin-like growth factor I (IGF-I). This was associated with a reduction in the P450 aromatase and P450scc (cholesterol side-chain cleavage cytochromes P450) (P450scc) protein levels but not those of 3?hydroxysteroid dehydrogenase (3?HSD) or steroidogenic acute regulatory protein (StAR) and with a decrease in IGF-I-induced IGF-I receptor and mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. Resistin treatment does not affect IGF-I-induced cell proliferation and basal steroidogenesis (there is no IGF-I or follicle-stimulating hormone stimulation). In the basal state, resistin rapidly stimulates Akt and MAPK ERK1/2 and p38 phosphorylation in primary human GCs. CONCLUSION(S): Resistin is present in human GCs and theca cells. It decreases P and E2 secretion, P450scc and P450 aromatase protein levels, and IGF-IR signaling in response to IGF-I in primary human GCs.
Serum resistin level is a predictor of ovarian response in in vitro fertilisation cycle. Chen YC et al. Objectives. To measure serum resistin levels in infertile women undergoing in vitro fertilisation-embryo transfer (IVF-ET), and to find any correlations between serum resistin levels and body weight, body mass index, the number of oocytes retrieved, and the outcome of IVF-ET. In addition, to assess whether there is any difference in serum resistin levels between infertility caused by polycystic ovary syndrome (PCOS) and infertility caused by other female factors. Methods. We designed a case-control study, and a total of 44 infertile women were enrolled. The blood samples for resistin measurement were collected on day 3 of the menstrual cycle prior to the administration of gonadotropin during in vitro fertilisation. These cases were then divided into 2 subgroups (PCOS group versus non-PCOS group) and a number of variables were measured and compared, including serum resistin levels. Results. Serum resistin levels were inversely correlated with the number of oocytes retrieved (r=-0.371, p=0.013). No significant correlation was found between serum resistin levels and body mass index or body weight, either in the whole group or in the 2 subgroups. Serum resistin levels in the non-PCOS group were significantly higher than in the PCOS group (p=0.049). Serum resistin levels in the non-PCOS group were inversely correlated to the number of oocytes retrieved (r=-0.386, p=0.039), but no similar correlation was found in the PCOS group. There was no correlation between serum resistin levels and fertility rate or clinical pregnancy rate in either subgroup. Conclusions. We observed a negative correlation between serum resistin levels and the number of oocytes retrieved during IVF. However, this phenomenon was only present in the non-PCOS group. This result suggests that serum resistin levels might be a good predictor of ovarian response in infertile women without PCOS during IVF. The role of serum resistin in response to inflammation caused by endometriosis or chronic pelvic infection, both of which are major causes of female infertility, should be examined in a further study.
Effect of resistin on granulosa and theca cell function in cattle. Spicer LJ et al. Resistin is an adipokine that has not been extensively studied in cattle but is produced by adipocytes in greater amounts in lactating versus non-lactating cattle. Seven experiments were conducted to determine the effect of resistin on proliferation, steroidogenesis, and gene expression of theca and granulosa cells from small (1-5mm) and/or large (8-22mm) cattle follicles. Resistin had no effect on IGF-I-induced proliferation of large-follicle theca cells or small-follicle granulosa cells, but decreased IGF-I-induced proliferation of large-follicle granulosa cells. Resistin weakly stimulated FSH plus IGF-I-induced estradiol production by large-follicle granulosa cells, but had no effect on IGF-I- or insulin-induced progesterone and androstenedione production by theca cells or progesterone production by granulosa cells of large follicles. In small-follicle granulosa cells, resistin attenuated the stimulatory effect of IGF-I on progesterone and estradiol production of small-follicle granulosa cells. RT-PCR measuring abundance of side-chain cleavage enzyme (CYP11A1), aromatase (CYP19A1), FSH receptor (FSHR) and LH receptor (LHCGR) mRNA in large- and small-follicle granulosa cells indicated that resistin reduced the stimulatory effect of IGF-I on CPY11A1 mRNA abundance in large-follicle granulosa cells but had no effect on CYP19A1, FSHR or LHCGR mRNA abundance in large- or small-follicle granulosa cells. Resistin had no effect on CYP11A1, CYP17A1 or LHCGR mRNA abundance in theca cells. These results indicate that resistin preferentially inhibits steroidogenesis of undifferentiated (small follicle) granulosa cells and inhibits proliferation of differentiated (large follicle) granulosa cells, indicating that the ovarian response to resistin is altered during follicular development./////Effects of resistin on porcine ovarian follicle steroidogenesis in prepubertal animals: an in vitro study. Rak-Mardy A A et al. BACKGROUND: Resistin was first reported to be an adipocyte-specific hormone, but recent studies have indicated a connection between resistin and reproductive function. However, it is not yet known if resistin is expressed by the ovary and if it can affect steroidogenesis in ovarian follicles from prepubertal pigs. METHODS: In this study, using real time PCR, immunoblotting, and ELISA, we quantified resistin expression and concentration in maturing ovarian follicles (small, 3--4 mm; medium, 4--5 mm; large, 6--7 mm) collected from prepubertal pigs. In addition, the dose-responsive effects of recombinant human resistin (0.1, 1, 10, and 100 ng/ml) on steroid hormone (i.e., progesterone [P4], androstendione [A4], testosterone [T], and estradiol [E2]) secretion in culture medium and steroidogenic enzyme (i.e., CYP11A1, 3betaHSD, CYP17A1, 17betaHSD, and CYP19A1) expression in ovarian follicles were determined. RESULTS: We observed that resistin gene and protein expression increased significantly (P < 0.05) during follicular growth, with large follicles expressing the highest level of this adipokine. Recombinant resistin also increased P4, A4, and T secretion by up-regulating the steady state levels of CYP11A1, 3betaHSD, CYP17A1, and 17betaHSD. Recombinant resistin had no effects on E2 secretion and CYP19A1 expression in ovarian follicles. CONCLUSION: Our results show resistin expression in ovarian follicles from prepubertal pigs for the first time. We also show that recombinant resistin stimulates steroidogenesis in ovarian follicles by increasing the expression of CYP11A1, 3betaHSD, CYP17A1, and 17betaHSD. The presence of resistin in the porcine ovary and its direct effects on steroidogenesis suggest that resistin is a new regulator of ovary function in prepubertal animals.
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Expression and effect of resistin on bovine and rat granulosa cell steroidogenesis and proliferation. Maillard V et al. Resistin, initially identified in adipose tissue and macrophages, was implicated in insulin resistance. Recently, its mRNA was found in hypothalamo-pituitary axis and rat testis, leading us to hypothesize that resistin may be expressed in ovary. Here, we determined in rat and cow: 1) the characterization of resistin in ovary by RT-PCR, immunoblotting and immunohistochemistry 2) the effects of recombinant resistin (10, 100, 333 and 667 ng/mL) +/- IGF1 (76 ng/mL) on steroidogenesis, proliferation and signaling pathways of granulosa cells (GC) measured by enzyme immunoassay, [3H]-thymidine incorporation and immunoblotting, respectively. We observed that resistin mRNA and protein were present in several bovine and rat ovarian cells. Nevertheless, only bovine GC abundantly expressed resistin mRNA and protein. Resistin treatment decreased basal, but not IGF1-induced progesterone (P<0.05; whatever the dose) and estradiol (P<0.005; for 10 and 333 ng/mL) production by bovine GC. In rat, resistin (10 ng/mL) increased basal and IGF1-induced progesterone secretion (P<0.0001), without effect on estradiol release. We found no effect of resistin on rat GC proliferation. Conversely in cow, resistin increased basal proliferation (P<0.0001; for 100 to 667 ng/mL) and decreased IGF1-induced proliferation of GC (P<0.0001; for 10 to 333 ng/mL) associated with a decrease in cyclin D2 protein level (P<0.0001). Finally, resistin stimulated Akt and p38-MAPK phosphorylation in both species, ERK1/2-MAPK phosphorylation in rat and had opposite effects on AMPK pathway (P<0.05). In conclusion, our results show that resistin is expressed in rat and bovine ovary. Furthermore, it can modulate granulosa cells functions in basal state or in response to IGF1 in vitro.
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