RESUMO
Bisphenol (BP) structural analogues of BPA are widely used. Previous studies showed similar effects of BPA and BPS on reproduction in several species including human. We hypothesised that the similar effects of several bisphenols (BPs) could accumulate in granulosa cells (GCs) and affects steroidogenesis. This study investigated the effects of seven BP analogues and their equimolar cocktail on human granulosa cells (hGC) and assessed BPA, BPS, BPF and BPAF level exposures in the follicular fluid of 277 women undergoing Assisted Reproductive Technology. The hGCs were recovered after women oocyte punctures and treated with the seven BP analogues (BPS, BPA, BPAF, BPF, BPAP, BPE and BPB) or their equimolar cocktail of 7 × 1.43 or 7 × 7.14 µM for each of the seven BPs, the sum of BPs reaching 10 ("∑BPs 10 µM"), or 50 µM ("∑BPs 50 µM"), respectively. Oestradiol and progesterone secretion, cell proliferation, viability and expression of steroidogenic enzymes were investigated. Progesterone secretion was decreased by 6 BPs 10 µM and the cocktail "∑BPs 10 µM", (-17.8 to -41.3%) and by all seven BPs 50 µM and "∑BPs 50 µM" (-21.8 to -84.2%). Oestradiol secretion was decreased only by 50 µM BPAF and BPAP (-37.8% and -44%, respectively), with corresponding decreases in CYP17A1 and CYP19A1 gene expression. Cellular proliferation was decreased after treatment with 50 µM BPAF (-32.2%), BPAP (-29%), BPB (-24%) and the equimolar cocktail "∑BPs 50 µM" (-33.1%). BPB (50 µM) and the cocktail "∑BPs 50 µM" increased HSD3B2 mRNA expression. At least one BP was detected in 64 of 277 (23.1%) women follicular fluids. Similar effects of the seven BPs or their cocktail were observed on progesterone secretion and/or on cell proliferation, suggesting cumulative effects of BPs. Our results highlight the urge to consider all BPs simultaneously and to further investigate the potential additive or synergistic effects of several BPs.
Assuntos
Compostos Benzidrílicos , Progesterona , Humanos , Feminino , Masculino , Compostos Benzidrílicos/farmacologia , Células da Granulosa , EstradiolRESUMO
BACKGROUND: Ovarian granulosa cells (GC) are essential for the development and maturation of a proper oocyte. GC are sensitive to endocrine disruptors, including bisphenol A (BPA) and its analogue bisphenol S (BPS), plasticisers present in everyday consumer products. BPA exhibits greater binding affinity for the membrane oestrogen receptor (GPER) than for the nuclear oestrogen receptors (ERα and ERß). Here, we analysed the effects of BPA and BPS on the steroidogenesis of ovine GC in vitro, as well as their early mechanisms of action, the ovine being a relevant model to study human reproductive impairment. Disruption of GC steroidogenesis might alter oocyte quality and consequently fertility rate. In addition, we compared the effects of a specific GPER agonist (G-1) and antagonist (G-15) to those of BPA and BPS. Ewe GC were cultured with BPA or BPS (10 or 50 µM) or G-1 (1 µM) and/or G-15 (10 µM) for 48 h to study steroidogenesis. RESULTS: Both BPA and BPS (10 µM) altered the secretion of progesterone, however, only BPS (10 µM) affected oestradiol secretion. RNA-seq was performed on GC after 1 h of culture with BPA or BPS (50 µM) or G-1 (10 µM), followed by real-time PCR analyses of differentially expressed genes after 12, 24 and 48 h of culture. The absence of induced GPER target genes showed that BPA and BPS did not activate GPER in GC after 1 h of treatment. These molecules exhibited mainly independent early mechanisms of action. Gene ontology analysis showed that after 1 h of treatment, BPA mainly disrupted the expression of the genes involved in metabolism and transcription, while BPS had a smaller effect and impaired cellular communications. BPA had a transient effect on the expression of CHAC1 (NOTCH signalling and oxidative balance), JUN (linked to MAPK pathway), NR4A1 (oestradiol secretion inhibition), ARRDC4 (endocytose of GPCR) and KLF10 (cell growth, differentiation and apoptosis), while expression changes were maintained over time for the genes LSMEM1 (linked to MAPK pathway), TXNIP (oxidative stress) and LIF (cell cycle regulation) after 12 and 48 h, respectively. CONCLUSION: In conclusion, although they exhibited similar effects, BPA and BPS impaired different molecular pathways in GC in vitro. New investigations will be necessary to follow the temporal changes of these genes over time, as well as the biological processes involved.
Assuntos
Células da Granulosa , Oócitos , Feminino , Ovinos , Animais , Humanos , Hormônios Esteroides Gonadais , EstradiolRESUMO
In mammals, the medio-basal hypothalamus (MBH) integrates photoperiodic and food-related cues to ensure timely phasing of physiological functions, including seasonal reproduction. The current human epidemics of obesity and associated reproductive disorders exemplifies the tight link between metabolism and reproduction. Yet, how food-related cues impact breeding at the level of the MBH remains unclear. In this respect, the sheep, which is a large diurnal mammal with a marked dual photoperiodic/metabolic control of seasonal breeding, is a relevant model. Here, we present a large-scale study in ewes (n = 120), which investigated the impact of food restriction (FRes) on the MBH transcriptome using unbiased RNAseq, followed by RT-qPCR. Few genes (~100) were impacted by FRes and the transcriptional impact was very modest (<2-fold increase or < 50% decrease for most genes). As anticipated, FRes increased expression of Npy/AgRP/LepR and decreased expression of Pomc/Cartpt, while Kiss1 expression was not impacted. Of particular interest, Eya3, Nmu and Dio2, genes involved in photoperiodic decoding within the MBH, were also affected by FRes. Finally, we also identified a handful of genes not known to be regulated by food-related cues (e.g., RNase6, HspA6, Arrdc2). In conclusion, our transcriptomics study provides insights into the impact of metabolism on the MBH in sheep, which may be relevant to human, and identifies possible molecular links between metabolism and (seasonal) reproduction.
Assuntos
Hipotálamo , Transcriptoma , Humanos , Animais , Ovinos , Feminino , Estações do Ano , Hipotálamo/metabolismo , Fotoperíodo , Reprodução/fisiologia , MamíferosRESUMO
Bisphenol S (BPS) affects terminal folliculogenesis by impairing steroidogenesis in granulosa cells from different species. Nevertheless, limited data are available on its effects during basal folliculogenesis. In this study, we evaluate in vitro the effects of a long-term BPS exposure on a model of basal follicular development in a mono-ovulatory species. We cultured ovine preantral follicles (180−240 µm, n = 168) with BPS (0.1 µM (possible human exposure dose) or 10 µM (high dose)) and monitored antrum appearance and follicular survival and growth for 15 days. We measured hormonal secretions (oestradiol (at day 13 [D13]), progesterone and anti-Müllerian hormone [D15]) and expression of key follicular development and redox status genes (D15) in medium and whole follicles, respectively. BPS (0.1 µM) decreased oestradiol secretion compared with the control (−48.8%, p < 0.001), without significantly impairing antrum appearance, follicular survival and growth, anti-Müllerian hormone and progesterone secretion and target gene expression. Thus, BPS could also impair oestradiol secretion during basal folliculogenesis as it is the case during terminal folliculogenesis. It questions the use of BPS as a safe BPA substitute in the human environment. More studies are required to elucidate mechanisms of action of BPS and its effects throughout basal follicular development.
RESUMO
Bisphenol A (BPA), a plasticizer and endocrine disruptor, has been substituted by bisphenol S (BPS), a structural analogue that had already shown adverse effects on granulosa cell steroidogenesis. The objective of this study was to assess the effect of chronic exposure to BPS, a possible endocrine disruptor, on steroid hormones in the ovary, oviduct and plasma using the ewe as a model. Given the interaction between steroidogenesis and the metabolic status, the BPS effect was tested according to two diet groups. Eighty adult ewes were allotted to restricted (R) and well-fed (WF) groups, that were further subdivided into two subgroups. Ewes were exposed to 50 µg BPS/kg/day in their diet (R50 and WF50 groups) or were unexposed controls (R0 and WF0 groups). After at least 3 months of BPS exposure, preovulatory follicular fluid, oviduct fluid and plasma were collected and steroid hormones were analyzed by gas chromatography coupled with tandem mass spectrometry (GC-MS/MS). A deleterious effect of restricted diet on the volume of oviduct fluid and numbers of pre-ovulatory follicles was observed. Exposure to BPS impaired estradiol concentrations in both follicular and oviduct fluids of well-fed ewes and progesterone, estradiol and estrone concentrations in plasma of restricted ewes. In addition, a significant interaction between metabolic status and BPS exposure was observed for seven steroids, including estradiol. In conclusion, BPS acts in ewes as an endocrine disruptor with differential actions according to metabolic status.
Assuntos
Disruptores Endócrinos , Animais , Disruptores Endócrinos/toxicidade , Estradiol , Feminino , Humanos , Oviductos/metabolismo , Fenóis , Progesterona/metabolismo , Ovinos , Sulfonas , Espectrometria de Massas em TandemRESUMO
Docosahexaenoic acid (DHA) is an n-3 polyunsaturated fatty acid (PUFA) that improves fertility by increasing membrane fluidity. Moreover, embryos produced by donor females supplied with n-3 PUFA did not show any difference in terms of the lipid profile after 7 days of culture. The present study aimed to investigate the effects of DHA (20 and 100 µM) coupled with carnosine (5 mg/mL), an antioxidant, during oocyte maturation and embryo development on the developmental and cryosurvival rates and the number of pluripotent cells. Free fatty acid receptor-4 (FFAR4), which is able to bind DHA, was visualised by immunostaining. The addition of DHA in the in vitro development (IVD) medium decreased the percentage of pluripotent SOX2 positive cells compared with the control (8.4% vs. 10.9%) without affecting the number of cells (196.7 vs. 191.6 cells) or the developmental (20.9% vs. 23.9% blastocysts rate on D7) and cryosurvival rates (86.3% vs 86.2%). Such a decrease in pluripotent cells, relevant to the differentiation of the first lineage within the inner cell mass, represents an improvement in the embryo quality. On the contrary, embryos without any pluripotent SOX2-positive cells would not be able to achieve gestation. Future studies should follow up these results by carrying out embryo transfers to assess the beneficial effects of DHA supplementation.
Assuntos
Ácidos Docosa-Hexaenoicos , Técnicas de Maturação in Vitro de Oócitos , Animais , Blastocisto , Bovinos , Criopreservação/veterinária , Ácidos Docosa-Hexaenoicos/farmacologia , Técnicas de Cultura Embrionária/veterinária , Desenvolvimento Embrionário , Feminino , Fertilização in vitro/veterinária , Técnicas de Maturação in Vitro de Oócitos/métodos , Técnicas de Maturação in Vitro de Oócitos/veterinária , OócitosRESUMO
Bisphenol A (BPA), an endocrine disruptor, has been replaced by structural analogues including bisphenol S (BPS). BPA and BPS exhibited similar effects regarding reproductive functions. Moreover, metabolic status and lipid metabolism are related to female fertility and could worsen BPS effects. The objective was to determine BPS in vivo effects on folliculogenesis and embryo production after chronic exposure through diet, and the influence of metabolic status in adult ewes. Sixty primiparous 2.5 year-old ewes, undergoing a restricted or well fed diet, were exposed to BPS (0, 4 or 50 µg/kg/day) for at least three months. After hormonal oestrus synchronisation and ovarian stimulation, ewes were subjected to ovum pick-up (OPU) procedures to collect immature oocytes, that underwent in vitro maturation, fertilisation and embryo production. Body weight, body condition score and plasma glucose were higher in well-fed compared to restricted ewes, while plasma NEFA was lower during the 4-5 months after the beginning of the diets. Plasma progesterone levels increased on day 5 before OPU session in well-fed compared to restricted ewes. No effect of BPS dose was observed on follicle population, plasma AMH levels and embryo production numbers and rates. However, a significant diet x BPS dose interaction was reported for cleaved embryos, > 4-cell embryos, blastocyst and early blastocyst numbers, and plasma triiodothyronine levels. Our study showed that a contrasted diet did not affect follicle population nor embryo production in adult ewes but could affect the quality and progesterone secretion of the corpus luteum. Chronic low BPS exposure had no effect on follicular population and oocyte competence. Nevertheless, the significant diet x dose interactions observed on embryo production suggest that BPS effect is modulated by metabolic status. Further studies are required to assess the risk of BPS exposure for public reproductive health.
Assuntos
Oócitos , Sulfonas , Animais , Dieta/veterinária , Embrião de Mamíferos , Feminino , Fenóis , OvinosRESUMO
Sexual reproduction requires the fertilization of a female gamete after it has undergone optimal development. Various aspects of oocyte development and many molecular actors in this process are shared among mammals, but phylogeny and experimental data reveal species specificities. In this chapter, we will present these common and distinctive features with a focus on three points: the shaping of the oocyte transcriptome from evolutionarily conserved and rapidly evolving genes, the control of folliculogenesis and ovulation rate by oocyte-secreted Growth and Differentiation Factor 9 and Bone Morphogenetic Protein 15, and the importance of lipid metabolism.
Assuntos
Evolução Biológica , Expressão Gênica/genética , Oócitos/crescimento & desenvolvimento , Animais , Feminino , MamíferosRESUMO
Bisphenol S (BPS) is a structural analog of the endocrine disruptor bisphenol A (BPA); it is the main BPA replacement in the plastics industry. Previous studies have shown that BPA and BPS exhibit similar effects on reproduction in fish and rodent species. BPS reportedly alters steroidogenesis in bovine granulosa cells. Luteinised granulosa cells collected from 59 women who were undergoing an in vitro fertilization procedure were cultured for 48 h in the presence or absence of BPS (10 nM, 100 nM, 1 µM, 10 µM or 50 µM). BPS exposure was investigated by assessing follicular fluids from these 59 women for their BPS content. Culture medium, cells, total messenger RNA (mRNA) and total protein extracted from the luteinised granulosa cells were examined for oestradiol and progesterone secretion, cellular proliferation, viability, gene expression, steroidogenic enzyme expression and cell signaling. BPS was measured in follicular fluids using mass spectrometry. Exposure of granulosa cells to 10 or 50 µM BPS for 48 h induced a 16% (p = 0.0059) and 64% (p < 0.0001) decrease, respectively, in progesterone secretion; 50 µM BPS decreased oestradiol secretion by 46% (p < 0.0001). Ten µM BPS also tended to reduce CYP11A1 protein expression by 37% (p = 0.0947) without affecting HSD3B1 and CYP19A1 expression. Fifty µM BPS increased ERRγ expression. Environmental levels of BPS (nanomolar range) did not induce changes in steroidogenesis in human granulosa cells. The effects of BPS were observed after only 48 h of BPS exposure. These acute effects might be similar to chronic effects of physiological BPS levels.
Assuntos
Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , Líquido Folicular/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células da Granulosa/metabolismo , Fenóis/farmacologia , Progesterona/biossíntese , Sulfonas/farmacologia , Células Cultivadas , Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Feminino , Líquido Folicular/efeitos dos fármacos , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/patologia , Humanos , Técnicas In VitroRESUMO
Bisphenols, plasticisers used in food containers, can transfer to food. Bisphenol A (BPA) has been described as an endocrine disruptor and consequently banned from the food industry in several countries. It was replaced by a structural analogue, Bisphenol S (BPS). BPA action on the steroidogenesis is one of the mechanisms underlying its adverse effects on the efficiency of female reproduction. This study aimed to determine whether BPS is a safe alternative to BPA regarding GC functions. Antral follicles (2-6 mm), of approximatively 1000 adult ewe ovaries, were aspired and GC purified. For 48 h, ovine GC were treated with BPA or BPS (from 1 nM to 200 µM) and the effects on cell viability, proliferation, steroid production, steroidogenic enzyme expression and signalling pathways were investigated. Dosages at and greater than 100 µM BPA and 10 µM BPS decreased progesterone secretion by 39% (P < 0.001) and 22% (P = 0.040), respectively. BPA and BPS 10 µM and previously mentioned concentrations increased oestradiol secretion two-fold (P < 0.001 and P = 0.082, respectively). Only 100 µM BPA induced a decrease (P < 0.001) in gene expression of the enzymes of steroidogenesis involved in the production of progesterone. BPA reduced MAPK3/1 phosphorylation and ESR1 and ESR2 gene expression, effects that were not observed with BPS. BPA and BPS altered steroidogenesis of ovine GC. Thus, BPS does not appear to be a safe alternative for BPA. Further investigations are required to elucidate BPA and BPS mechanisms of action.
Assuntos
Compostos Benzidrílicos/farmacologia , Disruptores Endócrinos/farmacologia , Estradiol/metabolismo , Células da Granulosa/efeitos dos fármacos , Folículo Ovariano/efeitos dos fármacos , Fenóis/farmacologia , Progesterona/metabolismo , Sulfonas/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Expressão Gênica/efeitos dos fármacos , Células da Granulosa/metabolismo , Folículo Ovariano/metabolismo , Fosforilação/efeitos dos fármacos , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Ovinos , Transdução de Sinais/efeitos dos fármacosRESUMO
INTRODUCTION: Bisphenol A (BPA) is a widespread compound in the plastic industry that is especially used to produce baby bottles, food packaging and metal cans. BPA, an endocrine disruptor, leads to alterations in reproductive function and therefore has been banned from the food industry. Unregulated BPA analogues, particularly Bisphenol S (BPS), have emerged and are now used in the plastic industry. Thus, this study aimed to examine the acute effects of low and environmental doses of BPS on ewe oocyte quality and developmental competence, and its mechanism of action, during in vitro maturation. METHODS: Ewe cumulus-oocyte complexes underwent in vitro maturation in the presence or absence of BPS (1 nM, 10 nM, 100 nM, 1 µM or 10 µM). Oocytes were then subjected to in vitro fertilisation and development. RESULTS: 1 µM BPS induced a 12.7% decrease in the cleavage rate (p = 0.004) and a 42.6% decrease in the blastocyst rate (p = 0.017) compared to control. The blastocyst rate reduction was also observed with 10 nM BPS. Furthermore, 10 µM BPS reduced the oocyte maturation rate, and 1 µM BPS decreased cumulus cell progesterone secretion. PR and AMH gene expression were reduced in cumulus cells. BPS induced a 5-fold increase in MAPK 3/1 activation (p = 0.04). CONCLUSIONS: BPS impaired ewe oocyte developmental competence. The data suggest that BPS might not be a safe BPA analogue. Further studies are required to elucidate its detailed mechanism of action.
Assuntos
Técnicas de Maturação in Vitro de Oócitos/métodos , Oócitos/efeitos dos fármacos , Oócitos/crescimento & desenvolvimento , Fenóis/farmacologia , Sulfonas/farmacologia , Animais , Compostos Benzidrílicos/antagonistas & inibidores , Compostos Benzidrílicos/farmacologia , Blastocisto/efeitos dos fármacos , Blastocisto/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células do Cúmulo/efeitos dos fármacos , Células do Cúmulo/metabolismo , Desenvolvimento Embrionário/efeitos dos fármacos , Desenvolvimento Embrionário/genética , Disruptores Endócrinos/farmacologia , Fertilização in vitro , Regulação da Expressão Gênica no Desenvolvimento , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Oócitos/metabolismo , Oogênese/efeitos dos fármacos , Fenóis/antagonistas & inibidores , Fosforilação , Progesterona/metabolismo , Ovinos , Sulfonas/antagonistas & inibidoresRESUMO
This work presents a systematic approach to study the conservation of genes between fruit flies and mammals. We have listed 971 Drosophila genes involved in female reproduction at the ovarian level and systematically looked for orthologs in the Ciona, zebrafish, coelacanth, lizard, chicken, and mouse. Depending on the species, the percentage of these Drosophila genes with at least one ortholog varies between 69% and 78%. In comparison, only 42% of all the Drosophila genes have an ortholog in the mouse genome (P < 0.0001), suggesting a dramatically higher evolutionary conservation of ovarian genes. The 177 Drosophila genes that have no ortholog in mice and other vertebrates correspond to genes that are involved in mechanisms of oogenesis that are specific to the fruit fly or the insects. Among 759 genes with at least one ortholog in the zebrafish, 73 have an expression enriched in the ovary in this species (RNA-seq data). Among 760 genes that have at least one ortholog in the mouse; 76 and 11 orthologs are reported to be preferentially and exclusively expressed in the mouse ovary, respectively (based on the UniGene expressed sequence tag database). Several of them are already known to play a key role in murine oogenesis and/or to be enriched in the mouse/zebrafish oocyte, whereas others have remained unreported. We have investigated, by RNA-seq and real-time quantitative PCR, the exclusive ovarian expression of 10 genes in fish and mammals. Overall, we have found several novel candidates potentially involved in mammalian oogenesis by an evolutionary approach and using the fruit fly as an animal model.
Assuntos
Cordados/genética , Drosophila melanogaster/genética , Oogênese/genética , Homologia de Sequência do Ácido Nucleico , Animais , Sequência de Bases , Evolução Biológica , Sequência Conservada , Feminino , Masculino , CamundongosRESUMO
BACKGROUND: Docosahexaenoic acid (DHA) is a n-3 polyunsaturated fatty acid (PUFA) belonging to a family of biologically active fatty acids (FA), which are known to have numerous health benefits. N-3 PUFAs affect reproduction in cattle, and notably directly affect follicular cells. In terms of reproduction in cattle, n-3 PUFA-enriched diets lead to increased follicle size or numbers. METHODS: The objective of the present study was to analyze the effects of DHA (1, 10, 20 and 50 µM) on proliferation and steroidogenesis (parametric and/or non parametric (permutational) ANOVA) of bovine granulosa cells in vitro and mechanisms of action through protein expression (Kruskal-Wallis) and signaling pathways (non parametric ANOVA) and to investigate whether DHA could exert part of its action through the free fatty acid receptor 4 (FFAR4). RESULTS: DHA (10 and 50 µM) increased granulosa cell proliferation and DHA 10 µM led to a corresponding increase in proliferating cell nuclear antigen (PCNA) expression level. DHA also increased progesterone secretion at 1, 20 and 50 µM, and estradiol secretion at 1, 10 and 20 µM. Consistent increases in protein levels were also reported for the steroidogenic enzymes, cytochrome P450 family 11 subfamily A member 1 (CYP11A1) and hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1 (HSD3B1), and of the cholesterol transporter steroidogenic acute regulatory protein (StAR), which are necessary for production of progesterone or androstenedione. FFAR4 was expressed in all cellular types of bovine ovarian follicles, and in granulosa cells it was localized close to the cellular membrane. TUG-891 treatment (1 and 50 µM), a FFAR4 agonist, increased granulosa cell proliferation and MAPK14 phosphorylation in a similar way to that observed with DHA treatment. However, TUG-891 treatment (1, 10 and 50 µM) showed no effect on progesterone or estradiol secretion. CONCLUSIONS: These data show that DHA stimulated proliferation and steroidogenesis of bovine granulosa cells and led to MAPK14 phosphorylation. FFAR4 involvement in DHA effects requires further investigation, even if our data might suggest FFAR4 role in DHA effects on granulosa cell proliferation. Other mechanisms of DHA action should be investigated as the steroidogenic effects seemed to be independent of FFAR4 activation.
Assuntos
Bovinos , Proliferação de Células/efeitos dos fármacos , Ácidos Docosa-Hexaenoicos/farmacologia , Células da Granulosa/efeitos dos fármacos , Animais , Feminino , Expressão Gênica , Células da Granulosa/citologia , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Visfatin and resistin appear to interfere with reproduction in the gonads, but their potential action at the hypothalamic-pituitary level is not yet known. The aim of the present study was to investigate the mRNA and protein expression of these adipokines in murine gonadotroph cells and to analyse the effects of different concentrations of recombinant mouse visfatin and resistin (0.01, 0.1, 1 and 10ngmL-1) on LH secretion and signalling pathways in LßT2 cells and/or in primary female mouse pituitary cells. Both visfatin and resistin mRNA and protein were found in vivo in gonadotroph cells. In contrast with resistin, the primary tissue source of visfatin in the mouse was the skeletal muscle, and not adipose tissue. Visfatin and resistin both decreased LH secretion from LßT2 cells after 24h exposure of cells (P<0.03). These results were confirmed for resistin in primary cell culture (P<0.05). Both visfatin (1ngmL-1) and resistin (1ngmL-1) increased AMP-activated protein kinase α phosphorylation in LßT2 cells after 5 or 10min treatment, up to 60min (P<0.04). Extracellular signal-regulated kinase 1/2 phosphorylation was transiently increased only after 5min resistin (1ngmL-1) treatment (P<0.01). In conclusion, visfatin and resistin are expressed in gonadotroph cells and they may affect mouse female fertility by regulating LH secretion at the level of the pituitary.
Assuntos
Gonadotrofos/metabolismo , Hormônio Luteinizante/metabolismo , Nicotinamida Fosforribosiltransferase/metabolismo , Resistina/metabolismo , Transdução de Sinais/fisiologia , Tecido Adiposo/metabolismo , Animais , Células Cultivadas , Camundongos , Músculo Esquelético/metabolismo , Nicotinamida Fosforribosiltransferase/genética , Fosforilação , Resistina/genéticaRESUMO
The objective of this study was to determine whether fish oil supplement has an effect on adipose tissue lipid profiles and gene expression in postpartum dairy cows. Holstein cows were supplemented with either long-chain n-3 polyunsaturated fatty acid (PUFA; protected fish oil) or control PUFA (n-6; toasted soybeans) for 2mo after calving (n=23 per diet). These cows showed no difference in milk production or metabolic parameters, but exhibited a tendency toward a decrease in early embryo mortality rate after artificial insemination. We hypothesized that, in addition to this effect, modifications in adipose tissue (AT) gene expression and lipid profiles would occur in response to diet. Subcutaneous AT samples were thus collected from the dewlaps of n-3 and n-6 dairy cows at 1mo antepartum, and 1wk, 2mo, and 5mo postpartum for the analysis of lipids and gene expression. Lipid profiles were obtained by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in both positive and negative modes. We found 37 lipid species in the 200 to 1,200 m/z range, which differed between the n-3 and control groups, suggesting that the n-3 supplement affected the lipid composition through the enrichment of lipids integrating long-chain PUFA from fish oil sources: eicosapentaenoic and docosahexaenoic acid. Moreover, a decrease in triacylglycerolipids was observed in AT of n-3 supplemented cows. The expression of 44 genes involved in fatty acid metabolism and the adipokine system was assessed by real-time reverse-transcription PCR. Hierarchical clustering, according to either postpartum stage or diet, enabled us to group genes exhibiting similar kinetic properties during lactation or by those that varied in similar ways after n-3 supplementation, respectively. Among the genes exhibiting a dietary effect, FABP4, LIPE, CD36, and PLIN1 were overexpressed in n-3 AT samples compared with the control, suggesting an increase in lipolysis due to n-3 supplementation, which was reflected on lipolytic activity at the protein level (i.e., protein expression of fatty acid binding protein 4, phosphorylated perilipin 1, and phosphorylated hormone-sensitive lipase). This increase in lipolysis is relevant to the decrease in triglycerides observed in these samples. Gene expression analyses between n-3 and control AT samples also suggested that the n-3 diet could modulate the secretory functions of AT, possibly by affecting adipokine expression; however, this has to be confirmed at the protein level.
Assuntos
Dieta/veterinária , Ácidos Graxos Ômega-3 , Tecido Adiposo/metabolismo , Animais , Bovinos , Suplementos Nutricionais , Ácidos Graxos , Feminino , Lactação , Leite/químicaRESUMO
The objective of this study was to determine the effect of a rumen-protected fish oil supplement on the production and reproduction variables in postpartum dairy cows. Holstein cows (n=46) were given a basal total mixed diet plus one PUFA supplement: n-3 (n-3; protected fish oil; 1% dry matter intake (DMI); n=23) or control (n-6; toasted soybeans; 1.8% DMI; n=23), in a switchback design over two consecutive lactations. Supplements were added to the diet between calving and 2 months after calving to assess the effect on growth and maturation of ovarian follicles from which ovulation occurred around the day of insemination. Body weight (BW), milk yield (MY) and composition, dry matter intake (DMI), energy balance (EB), subcutaneous fat thickness, plasma fatty acid composition, plasma nonesterified fatty acids (NEFA), glucose and urea concentrations, follicular activity, embryo mortalities and fertility (conception rate after first AI, AI1) were assessed. BW, MY, DMI, plasma NEFA, glucose and urea were unaffected by the diet. There was a trend of an increased number of large follicles (diameter≥10mm) with the n-3 dietary supplementation (P=0.06) and a decrease in infertility or early embryo mortality rate 21 days after AI, 13.5% in the n-3 compared with 38.8% in the n-6 group (P=0.09), with no effect on the conception rate at 35d or 90d after AI1. These data suggest that the effect seen on ovarian variables is not associated with an effect on production and metabolic variables and is specific to n-3 PUFA supplementation. Further studies are necessary to determine whether DHA or EPA enhances fertility in lactating dairy cattle.
Assuntos
Ração Animal/análise , Bovinos/fisiologia , Dieta/veterinária , Ácidos Graxos Ômega-3/farmacologia , Lactação/efeitos dos fármacos , Reprodução/efeitos dos fármacos , Fenômenos Fisiológicos da Nutrição Animal , Animais , Composição Corporal , Bovinos/sangue , Suplementos Nutricionais , Ácidos Graxos Ômega-3/administração & dosagem , Ácidos Graxos Ômega-3/química , Feminino , Óleos de Peixe/administração & dosagem , Óleos de Peixe/química , Leite/química , Gordura Subcutânea/efeitos dos fármacos , Gordura Subcutânea/fisiologiaRESUMO
In mammals, oocytes develop inside the ovarian follicles; this process is strongly supported by the surrounding follicular environment consisting of cumulus, granulosa and theca cells, and follicular fluid. In the antral follicle, the final stages of oogenesis require large amounts of energy that is produced by follicular cells from substrates including glucose, amino acids and fatty acids (FAs). Since lipid metabolism plays an important role in acquiring oocyte developmental competence, the aim of this study was to investigate site-specificity of lipid metabolism in ovaries by comparing lipid profiles and expression of FA metabolism-related genes in different ovarian compartments. Using MALDI Mass Spectrometry Imaging, images of porcine ovary sections were reconstructed from lipid ion signals for the first time. Cluster analysis of ion spectra revealed differences in spatial distribution of lipid species among ovarian compartments, notably between the follicles and interstitial tissue. Inside the follicles analysis differentiated follicular fluid, granulosa, theca and the oocyte-cumulus complex. Moreover, by transcript quantification using real time PCR, we showed that expression of five key genes in FA metabolism significantly varied between somatic follicular cells (theca, granulosa and cumulus) and the oocyte. In conclusion, lipid metabolism differs between ovarian and follicular compartments.
RESUMO
In dairy cows, lipids are essential to support energy supplies for all biological functions, especially during early lactation. Lipid metabolism is crucial for sustaining proper reproductive function. Alteration of lipid metabolism impacts follicular development and affects oocyte developmental competence. Indeed, nonesterified fatty acids are able to decrease granulosa cell (GC) proliferation and affect estradiol synthesis, thus potentially affecting follicular growth and viability. The objective of this study was to assess the impact of lipid metabolism on bovine GCs, through the use of the lipid metabolism inhibitors etomoxir, an inhibitor of fatty acid (FA) oxidation through inhibition of carnitine palmitoyl transferase 1 (CPT1), and C75, an inhibitor of FA synthesis through inhibition of fatty acid synthase. We showed that etomoxir and C75 significantly inhibited DNA synthesis in vitro; C75 also significantly decreased progesterone synthesis. Both inhibitors significantly reduced AMPK (5' adenosine monophosphate-activated protein kinase) and acetyl-CoA carboxylase phosphorylation. Etomoxir also affected the AKT (protein kinase B) signaling pathway. Combined, these data suggest that both FA oxidation and synthesis are important for the bovine GCs to express a proliferative and steroidogenic phenotype and, thus, for sustaining follicular growth. Despite these findings, it is important to note that the changes caused by the inhibitors of FA metabolism on GCs in vitro are globally mild, suggesting that lipid metabolism is not as critical in GCs as was observed in the oocyte-cumulus complex. Further studies are needed to investigate the detailed mechanisms by which lipid metabolism interacts with GC functions.
Assuntos
Bovinos/fisiologia , Proliferação de Células/fisiologia , Células da Granulosa/metabolismo , Metabolismo dos Lipídeos , Progesterona/biossíntese , 4-Butirolactona/análogos & derivados , 4-Butirolactona/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Compostos de Epóxi/farmacologia , Feminino , Regulação da Expressão Gênica/fisiologia , Células da Granulosa/citologia , Células da Granulosa/fisiologia , Oxazinas , Transdução de Sinais , Coloração e RotulagemRESUMO
Oocyte meiotic maturation requires energy from various substrates including glucose, amino acids, and lipids. Mitochondrial fatty acid (FA) ß-oxidation (FAO) in the oocyte is required for meiotic maturation, which is accompanied by differential expression of numerous genes involved in FAs metabolism in surrounding cumulus cells (CCs) in vivo. The objective was to elucidate components involved in FAs metabolism in CCs during oocyte maturation. Twenty-seven genes related to lipogenesis, lipolysis, FA transport, and FAO were chosen from comparative transcriptome analysis of bovine CCs before and after maturation in vivo. Using real-time PCR, 22 were significantly upregulated at different times of in vitro maturation (IVM) in relation to oocyte meiosis progression from germinal vesicle breakdown to metaphase-II. Proteins FA synthase, acetyl-coenzyme-A carboxylase, carnitine palmitoyltransferase, perilipin 2, and FA binding protein 3 were detected by Western blot and immunolocalized to CCs and oocyte cytoplasm, with FA binding protein 3 concentrated around oocyte chromatin. By mass spectrometry, CCs lipid profiling was shown to be different before and after IVM. FAO inhibitors etomoxir and mildronate dose-dependently decreased the oocyte maturation rate in vitro. In terms of viability, cumulus enclosed oocytes were more sensitive to etomoxir than denuded oocytes. In CCs, etomoxir (150 µM) led to downregulation of lipogenesis genes and upregulated lipolysis and FAO genes. Moreover, the number of lipid droplets decreased, whereas several lipid species were more abundant compared with nontreated CCs after IVM. In conclusion, FAs metabolism in CCs is important to maintain metabolic homeostasis and may influence meiosis progression and survival of enclosed oocytes.
Assuntos
Células do Cúmulo/metabolismo , Ácidos Graxos/química , Oócitos/citologia , Oxigênio/química , Transporte Ativo do Núcleo Celular , Animais , Bovinos , Sobrevivência Celular , Análise por Conglomerados , Ácidos Graxos/biossíntese , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Lipogênese/genética , Mitocôndrias/metabolismo , Oogênese/fisiologia , Ovário/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
BACKGROUND: In mammals, the Tribbles family includes widely expressed serine-threonine kinase-like proteins (TRIB1, TRIB2 and TRIB3) that are involved in multiple biological processes including cell proliferation and fatty acid (FA) metabolism. Our recent studies highlighted the importance of FA metabolism in cumulus cells (CC) during oocyte maturation in vertebrates and reported a higher TRIB1 expression in CC surrounding in vivo mature oocytes as compared to immature ooocytes in mice and cows. The objective was to investigate Tribbles expression patterns in bovine CC during in vitro maturation (IVM) and to examine their roles in the cumulus-oocyte complex. METHODS: Tribbles gene expression was analyzed in bovine and murine CC using quantitative RT-PCR. Proteins were detected using Western blot and intracellular localization was assessed by immunofluorescence. Bovine COCs were treated with etomoxir, an inhibitor of FA oxidation (FAO) which blocks CPT1 activity, during 6 h and 18 h IVM. Oocyte meiotic stage was assessed and expression of Tribbles and lipid metabolism genes was quantified in CC. RESULTS AND DISCUSSION: TRIB1 and TRIB3 were more strongly expressed whereas TRIB2 was less expressed in CC surrounding the oocytes from preovulatory follicles than in CC of immature ones. In CC, Tribbles were located in the cytoplasm and nucleus; in mitotic cells TRIB2 and TRIB3 were detected in the spindle. In the oocyte, Tribbles proteins were detected in the ooplasm; also TRIB2 and TRIB3 were more accumulated in the germinal vesicle. In bovine CC, expression of TRIB1 and TRIB3 was transiently increased at a time preceding oocyte meiosis resumption in vitro. Treatment with etomoxir (150 µM) during IVM resulted in a significant reduction of oocyte maturation rate and decreased MAPK3/1 phosphorylation in the oocytes. In CC, 18 h IVM of etomoxir treatment significantly increased expression of TRIB1, TRIB3, CPTA1 (enzyme regulating FA entry in mitochondria for FAO) and CD36 (thrombospondin receptor involved in FA transport). Under the same conditions, expression of TRIB2 and ACACA (Acetyl coenzyme A carboxylase involved in FA synthesis) decreased in CC. All considered, Tribbles family members may be involved in cell proliferation and in FAO signaling in CC and participate in oocyte meiotic resumption regulation.