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1.
J Anim Sci ; 98(9)2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32901281

RESUMO

Bovine twin birth is associated with detriments, including increased embryo/fetal losses, malpresentation, and dystocia. Incidence of these is lessened in bilateral compared with unilateral twin pregnancy. This study was undertaken to assess the use of follicular ablation by aspiration to create bilateral twin pregnancies in females with genetic potential for ~3.5 ovulations per cycle (Trio allele carriers). In experiment 1, carriers (n = 30) and noncarriers (n = 10) were synchronized for ovulation and timed artificial insemination (TAI). Follicles (>5 mm) in excess of one per ovary were aspirated ~16 h preceding TAI. Follicle count for females with follicles on only one ovary was reduced to two. Blood was sampled 2 wk post-TAI to assess progesterone (P4) concentrations; embryo count was determined by ultrasound 6 wk post-TAI. Circulating P4 concentration post-TAI was significantly (P < 0.001) associated with both genotype and subsequent pregnancy status (pregnant noncarriers: 7.06 ± 0.68 ng/mL; pregnant carriers: 5.54 ± 0.55 ng/mL; nonpregnant noncarriers: 5.22 ± 1.05 ng/mL; nonpregnant carriers: 3.13 ± 0.42 ng/mL). Experiment 2 was undertaken to offset the negative effects of follicular aspiration on subsequent P4 concentration observed in experiment 1. Carriers (n = 38) and noncarriers (n = 32) were submitted to TAI and follicle ablation as described for experiment 1. Additionally, accessory corpora lutea (CL) were induced in carriers by the administration of human chorionic gonadotropin (carriers) at day 6 post-TAI. Consequently, P4 concentration post-TAI was significantly (P < 0.05) associated with subsequent pregnancy status (pregnant: 8.48 ± 0.61 ng/mL; nonpregnant: 6.70 ± 0.63 ng/mL) but not with genotype (carrier: 8.01 ± 0.59 ng/mL; noncarrier: 7.17 ± 0.64 ng/mL). Embryo number was greater in carriers (exp. 1: 1.64 ± 0.81; exp 2: 1.45 ± 0.09) vs. noncarriers (1.00 ± 0.00, both experiments). Single, twin, and triplet pregnancies occurred in carriers in experiment 1, whereas multiples in experiment 2 were limited to twin pregnancies. Genotype effects on pregnancy rate were not significant (P > 0.10) in either experiment. Results suggest that follicular ablation to create bilateral twin pregnancies in Trio carriers is feasible but requires the induction of accessory CL to offset the negative effects of follicular aspiration on subsequent P4 concentration and associated fertility outcomes.


Assuntos
Bovinos/genética , Fertilidade/genética , Ovulação/genética , Progesterona/análise , Alelos , Animais , Bovinos/fisiologia , Gonadotropina Coriônica/administração & dosagem , Corpo Lúteo/efeitos dos fármacos , Sincronização do Estro/efeitos dos fármacos , Feminino , Genótipo , Heterozigoto , Humanos , Inseminação Artificial/veterinária , Tamanho da Ninhada de Vivíparos , Folículo Ovariano/efeitos dos fármacos , Gravidez , Taxa de Gravidez
2.
J Dairy Sci ; 103(7): 6600-6611, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32359997

RESUMO

The objectives of this study were to evaluate the use of a qualitative on-farm milk progesterone test to predict non-pregnancy in dairy cows. Lactating Jersey cows (n = 752) were subjected to the 5-d Cosynch-72 protocol for timed artificial insemination (AI; d -8 GnRH, d -3 and -2 PGF2α, d 0 GnRH and timed AI). Milk was sampled on d -3, 0, 7, and 28 relative to timed AI, and progesterone concentrations were assessed using a lateral flow immunochromatographic test. Samples were classified into 3 groups indicative of high (hP4; test line not visible or lighter than reference), intermediate (iP4; test line similar to reference), and low (lP4; test line darker than reference) progesterone concentrations. Blood was sampled from a subset of cows (n = 50) on d -3, 0, 7, and 28 relative to timed AI, and plasma progesterone concentrations were determined by RIA. Cows were observed daily for signs of estrus based on removal of tail paint. Pregnancy was diagnosed by ultrasonography on d 34 and 62 after AI. Plasma progesterone concentrations across all time points were greater for hP4 (3.13 ± 0.20 ng/mL) followed by iP4 (1.12 ± 0.27 ng/mL) and lP4 (0.38 ± 0.23 ng/mL). Cows in lP4 on d -3 had lesser pregnancy per AI (P/AI) compared with iP4 and hP4 (17.4, 38.3, and 37.2%, respectively). For measurements performed on the day of AI (d 0), lP4 cows had greater P/AI compared with hP4 and iP4 (34.8, 0.0, and 15.6%, respectively), and the risk of pregnancy loss tended to be greater for iP4 compared with lP4. Cows in lP4 on d 7 after AI had lesser P/AI than those in iP4 and hP4 (12.0, 34.0, and 37.7%, respectively). Cows classified as lP4 on d 28 had the least P/AI on d 62 followed by iP4 and then hP4 (0.8, 9.2, and 59.4%, respectively) and were at the greatest risk for pregnancy loss (lP4 = 74.6%, iP4 = 8.4%, hP4 = 7.1%). Sensitivity and specificity to predict non-pregnancy on d 62 were 0.86 and 0.32 (d -3), 0.95 and 0.15 (d 0), 0.93 and 0.23 (d 7), and 0.99 and 0.53 (d 28), respectively. On-farm milk progesterone profiling using a lateral flow immunochromatographic test was able to identify cows without functional corpus luteum and to predict fertility outcomes following timed AI.


Assuntos
Bovinos/fisiologia , Fertilidade , Hormônio Liberador de Gonadotropina/administração & dosagem , Leite/química , Progesterona/análise , Animais , Corpo Lúteo/metabolismo , Estro , Sincronização do Estro , Fazendas , Feminino , Inseminação Artificial/veterinária , Lactação , Gravidez
3.
Food Chem Toxicol ; 138: 111204, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32081729

RESUMO

A new method, using liquid chromatography coupled to mass spectrometry (LC-MS/MS) for the detection of fourteen natural and synthetic hormones in muscles, was validated in other bovine matrices (liver, kidney, bile and hair) according to the Decision Commission 2002/657/EC. As result, this method demonstrates good linearity (R2 > 0.99) as well as accuracy with coefficients of variation for repeatability and reproducibility lower than 23%. Moreover, the values of decision limit (CCα) and detection capability (CCß) were determined indicating values ranging from 0.13 to 0.86 µg/kg and 0.25-1.72 µg/k for the majority of analytes. Recovery rate in the different matrices varied from 51.5 to 107%. Indeed, this method has been successfully applied to detect anabolic hormones in eighty-eight samples (muscle, liver, kidney, and bile) collected from different local slaughterhouses. Results showed that progesterone was found in 30 samples at concentrations ranging from 0.11 to 11.7 µg/kg, while testosterone was detected in 34 samples at concentrations ranging from 0.5 to 9.52 µg/kg. All bile samples contain epi-testosterone at concentration ranging from 0.89 to 280 µg/kg. These obtained data were used to calculate the estimated daily intake, hazard quotient and hazard index as exposure assessment.


Assuntos
Cromatografia Líquida de Alta Pressão , Espectrometria de Massas em Tandem , Animais , Bile/química , Bovinos , Cabelo/química , Rim/química , Limite de Detecção , Fígado/química , Músculos/química , Progesterona/análise , Reprodutibilidade dos Testes , Medição de Risco , Sensibilidade e Especificidade , Testosterona/análise
4.
Sci Rep ; 10(1): 2954, 2020 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-32075989

RESUMO

The blubber steroid hormone profiles of 52 female humpback whales migrating along the east coast of Australia were investigated for seasonal endocrine changes associated with reproduction. Individuals were randomly sampled during two stages of the annual migration: before reaching the breeding grounds (northward migration; June/July), and after departing from the breeding grounds (southward migration; September/October). Assignment of reproductive status of the sampled individuals was based on season, single-hormone ranks and multi-variate analysis of the hormonal profiles. High concentrations of progesterone (>19 ng/g, wet weight), recognised as an indicator of pregnancy in this species, were only detected in one sample. However, the androgens, testosterone and androstenedione were measured in unusually high concentrations (1.6-12 and 7.8-40 ng/g wet weight, respectively) in 36% of the females approaching the breeding grounds. The absence of a strong accompanying progesterone signal in these animals raises the possibility of progesterone withdrawal prior to parturition. As seen with other cetacean species, testosterone and androstenedione could be markers of near-term pregnancy in humpback whales. Confirmation of these androgens as alternate biomarkers of near-term pregnancy would carry implications for improved monitoring of the annual fecundity of humpback whales via non-lethal and minimally invasive methods.


Assuntos
Androstenodiona/análise , Jubarte/fisiologia , Testes de Gravidez/métodos , Gordura Subcutânea/química , Testosterona/análise , Animais , Biomarcadores/análise , Feminino , Gravidez , Progesterona/análise , Estações do Ano
5.
Am J Perinatol ; 37(6): 562-569, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31891958

RESUMO

OBJECTIVE: Preterm delivery following fetoscopic laser surgery (FLS) of twin-twin transfusion syndrome (TTTS) is associated with severe perinatal morbidity and mortality. The role of steroid hormones in amniotic fluid (AF) after FLS remains unknown. STUDY DESIGN: A prospective cohort study of consecutive case series of FLS for TTTS was performed from April 2012 to February 2017. Cases were divided into early (≤27 weeks) spontaneous preterm delivery (ED) and late delivery (LD; ≥34 weeks) following FLS and compared. AF supernatants were assessed for protein, estradiol, progesterone and cortisol levels (using the ELISA kit), and normalized to total protein levels to adjust for dilution. RESULTS: A total of 294 consecutive cases of FLS for TTTS in monochorionic-diamniotic twins were performed during the study period. AF was available in 44 ED patients and 50 LD patients. On logistic regression, ED was associated with higher normalized progesterone levels (odds ratio [OR]: 1.25; 95% confidence interval [CI]: 1.12-1.41), lower normalized cortisol (OR: 0.78; 95% CI: 0.64-0.96), and higher estradiol levels (OR: 1.3; 95% CI: 1.03-1.63). CONCLUSION: Elevated AF normalized progesterone and estradiol, and lower normalized cortisol levels were associated with ED. This novel finding requires further exploration to establish the molecular mechanism operational in pregnancies complicated by TTTS to potentially prevent early preterm birth after fetal surgery.


Assuntos
Líquido Amniótico/química , Transfusão Feto-Fetal , Nascimento Prematuro , Esteroides/análise , Adulto , Estradiol/análise , Estrogênios/análise , Feminino , Humanos , Hidrocortisona/análise , Modelos Logísticos , Gravidez , Resultado da Gravidez , Progesterona/análise , Estudos Prospectivos , Proteínas/análise
6.
Horm Behav ; 119: 104647, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31778719

RESUMO

Relatively little is known about the effects of endogenous and exogenous steroid hormones on ecologically relevant behavioral and cognitive phenotypes in women, such as emotion recognition, despite the widespread use of steroid hormone-altering hormonal contraceptives (HCs). Though some previous studies have examined the effect of HC use, estradiol, progesterone, and testosterone on emotion recognition in women, they have been limited by cross-sectional designs, small sample sizes (total n < 100), and compromised statistical power to detect significant effects. Using data from two test sessions in a large sample of naturally cycling women (NC; n = 192) and women on HCs (n = 203), we found no group differences in emotion recognition; further, the lack of group differences in emotion recognition was not modulated by item difficulty or emotional valence. Among NC women who provided saliva samples across two sessions that were assayed for estradiol and progesterone concentrations, we found no compelling evidence across models that between-subject differences and within-subject fluctuations in these ovarian hormones predicted emotion recognition accuracy, with the exception that between-subjects estradiol negatively predicted emotion recognition for emotions of neutral valence (p = .042). Among HC women who provided saliva samples across two sessions that were assayed for testosterone, we found no compelling evidence that between-subjects differences and within-subject fluctuations in testosterone predicted emotion recognition accuracy. Overall, our analyses provide little support for the idea that circulating endogenous or exogenous ovarian hormones influence emotion recognition in women.


Assuntos
Anticoncepcionais Orais Hormonais/farmacologia , Inteligência Emocional/efeitos dos fármacos , Hormônios Esteroides Gonadais/metabolismo , Reconhecimento Psicológico/efeitos dos fármacos , Adulto , Estudos Transversais , Inteligência Emocional/fisiologia , Emoções , Estradiol/análise , Estradiol/metabolismo , Feminino , Hormônios Esteroides Gonadais/análise , Humanos , Ovário/efeitos dos fármacos , Ovário/metabolismo , Progesterona/análise , Progesterona/metabolismo , Reconhecimento Psicológico/fisiologia , Saliva/química , Saliva/metabolismo , Testosterona/análise , Testosterona/metabolismo , Adulto Jovem
7.
Gen Comp Endocrinol ; 287: 113346, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31790656

RESUMO

Yolk hormones are substances which transmit non-genetic factors from the mother to the next generation. The systematic changes of yolk hormone concentrations within asynchronously hatching clutches have been interpreted as a means to adaptively shape the offspring's phenotype. However, in synchronously hatching clutches the role of yolk hormones is less understood. We investigated whether seasonal changes between eggs in the yolk hormones testosterone (Testo), progesterone (Prog) and corticosterone (Cort) also occur in the grey partridge, a synchronously hatching precocial species without direct food competition between siblings. Specifically we asked whether yolk hormone concentrations systematically vary with season and whether they affect the offspring's hatching mass, mass gain, circulating baseline and stress-induced Cort. Additionally, we investigated the effect of genetic background and food availability on yolk hormone concentrations by subjecting grey partridge hens of two strains (wild and domesticated) to two different feeding regimes (predictable vs. unpredictable feeding) during egg laying. We hypothesized that egg hormone concentrations change over the season, but breeding in captivity over many generations and ad libitum food access could have resulted in domestication effects which abolished potential seasonal effects. Results showed that progressing season had a strong positive effect on yolk Prog and yolk Testo, but not on yolk Cort. Feeding regimes and strain had no effect on yolk hormones. Offspring mass and mass gain increased and baseline Cort decreased with progressing season. In addition, yolk Testo correlated positively with offspring mass gain and negatively with baseline Cort, while yolk Prog had a positive correlation with baseline Cort. Strain and feeding regimes of the mother had no effect on offspring traits. In conclusion, grey partridge chicks hatching late in the season might benefit from the increased concentrations of the growth-stimulating yolk Testo and by this catch-up in development. Hence, yolk hormone concentration could adaptively shape the offspring phenotype in a precocial species.


Assuntos
Gema de Ovo/química , Galliformes , Hormônios/análise , Herança Materna/fisiologia , Efeitos Tardios da Exposição Pré-Natal , Animais , Corticosterona/análise , Corticosterona/metabolismo , Gema de Ovo/metabolismo , Feminino , Galliformes/metabolismo , Hormônios/metabolismo , Oviposição/fisiologia , Fenótipo , Gravidez , Efeitos Tardios da Exposição Pré-Natal/metabolismo , Progesterona/análise , Progesterona/metabolismo , Estações do Ano , Testosterona/análise , Testosterona/metabolismo
8.
Biosens Bioelectron ; 150: 111876, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31748195

RESUMO

Preparation and electrochemical interrogation of a novel redox active progesterone derivative progesterone thiosemicarbazone (PATC) is presented here together with an investigation into its suitability as conjugate in progesterone hormone immunosensing. PATC synthesis involved a condensation reaction between progesterone acetate and thiosemicarbazone hydrochloride. Voltammetric and pulse techniques confirmed the redox behaviour of the new compound with concentration and scan rate dependant irreversible behaviour evident at glassy carbon and gold transducers - ko (standard heterogeneous rate constant) was 2.56 × 10-3 cm2/s (ν = 100 mV/s in non-aqeuous media). Bioaffinity studies towards anti-progesterone antibodies involved a competitive ELISA format (optical) which confirmed recognition of the new progesterone derivative. Electrochemical impedance spectroscopy was employed as an interrogation technique in order to establish optimum binding and surface conditions for progesterone antigen-antibody interaction with the assistance of a redox probe (potassium hexacyanoferrate).


Assuntos
Anticorpos Imobilizados/química , Técnicas Biossensoriais/métodos , Progesterona/análise , Carbono/química , Espectroscopia Dielétrica/métodos , Ensaio de Imunoadsorção Enzimática , Ferricianetos/química , Ouro/química , Imunoensaio/métodos , Oxirredução , Progesterona/análogos & derivados , Transdutores
9.
Int J Eat Disord ; 53(2): 296-301, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31846101

RESUMO

OBJECTIVE: Research indicates a link between ovarian hormones and eating pathology, suggesting that some women with an eating disorder may be ovarian hormone sensitive. Using premenstrual symptoms (PMS) as an indirect measure of ovarian hormone sensitivity, we investigated the association between 11 PMS domains and four core eating disorder symptoms: body dissatisfaction, binge eating, purging, and restriction. METHOD: Participants were young adult women (N = 455) who completed an online survey. PMS were assessed using the Daily Record of Severity of Problems and eating pathology with the Eating Pathology Symptoms Inventory. Pearson correlations were calculated between PMS domains and eating disorder symptoms followed by a stepwise regression to create a more refined model for each eating disorder symptom, including relevant covariates. RESULTS: Significant correlations between a majority of eating disorder symptoms and PMS emerged (r's = .13-.37; p < .01). Backward regression revealed significant PMS domain predictors for each symptom. The final models captured a small-to-moderate amount of variance for each eating disorder symptom (R2 = 0.06-0.25). DISCUSSION: Women who experience physical and psychological PMS may be at risk for eating disorder symptoms; PMS could be a marker of ovarian hormone sensitivity in women at risk for an eating disorder. Future studies should address mechanisms underlying this association.


Assuntos
Biomarcadores/sangue , Estradiol/sangue , Transtornos da Alimentação e da Ingestão de Alimentos/complicações , Síndrome Pré-Menstrual/complicações , Progesterona/sangue , Adulto , Estradiol/análise , Feminino , Humanos , Progesterona/análise , Inquéritos e Questionários , Adulto Jovem
10.
Biosens Bioelectron ; 150: 111921, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31818754

RESUMO

Upconversion nanoparticles (UCNPs) are promising materials for biological applications based on luminescence resonance energy transfer (LRET). In contrast to classical RET donors such as quantum dots, gold nanoparticles, UCNPs can emit near-infrared (NIR) upon the NIR irradiation, which provides enhanced signal-to-noise due to strong penetration and low autofluorescence in the NIR region known as the diagnostic window. Here we report the first efficient NIR-to-NIR signal-based LRET system for the detection of progesterone, chosen as a proof-of-concept target, via homogeneous competitive immunoassay. To enhance the efficiency of LRET, we constructed inert-core/active-shell/inert-ultrathin shell UCNPs (NaYF4@NaYF4:Yb,Tm@NaYF4) as an LRET donor and a compact progesterone/horseradish peroxidase/IRdyeQC-1 (P-HRP-dyes) complex as an LRET acceptor. The designed donor and acceptor showed significantly improved LRET efficiencies (95% and 85% for donor and acceptor, respectively) compared with conventional donor and acceptor (70% and 50%, respectively). Using the developed NIR-to-NIR LRET system, progesterone was successfully detected with a background-free signal and low limit of detection (1.36 pg/ml in ten-fold diluted human serum). We believe that the efficient NIR-to-NIR signal-based LRET system developed here has potential as a simple probe for homogeneous competitive immunoassay, with the ability to rapidly detect biomarkers.


Assuntos
Anticorpos Imobilizados/química , Técnicas Biossensoriais/métodos , Fluoretos/química , Substâncias Luminescentes/química , Medições Luminescentes/métodos , Progesterona/sangue , Ítrio/química , Transferência de Energia , Humanos , Imunoensaio/métodos , Raios Infravermelhos , Progesterona/análise
11.
Theriogenology ; 142: 376-383, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31708192

RESUMO

In many mammalian species, corpus luteum derived progesterone (P4) is the main functional gestagen during the estrous cycle and pregnancy. P4 can be metabolized into various metabolites, of which some are biologically active. While some metabolites target the classical nuclear progesterone receptor (PR), neurosteroids bind the receptors of type A γ-aminobutyric acid (GABAA-r) in the brain. According to the position of reduction within the molecule, metabolites of P4 can be characterized into C20-reduced progestogens (20α-dihydroprogesterone (20α-DHP) and 20ß-dihydroprogesterone (20ß-DHP)), C3-reduced progestogens (3α-dihydroprogesterone (3α-DHP) and 3ß-dihydroprogesterone (3ß-DHP)), 5α-reduced progestogens (5α-dihydroprogesterone (5α-DHP), allopregnanolone and isopregnanolone) and 5ß-reduced progestogens (5ß-dihydroprogesterone (5ß-DHP), pregnanolone and epipregnanolone). We questioned whether the reduced progestogens are present in bovine plasma during the estrous cycle and whether their profiles differed from the profile of the common precursor P4 around the time of luteolysis. The analytes were monitored in plasma samples using liquid chromatography mass spectrometry (LC-MS). While progestogens lagged behind the drop of P4 at luteolysis, they followed the profile of P4 during the estrous cycle. The abundance of P4 was predominant followed by allopregnanolone, pregnanolone, epipregnanolone and 20ß-DHP. Further studies will need to focus particularly on the period around luteolysis.


Assuntos
Análise Química do Sangue , Bovinos/sangue , Ciclo Estral/sangue , Progestinas/sangue , Animais , Análise Química do Sangue/métodos , Análise Química do Sangue/veterinária , Cromatografia Líquida/veterinária , Feminino , Progesterona/análise , Progesterona/sangue , Progestinas/análise , Espectrometria de Massas em Tandem/veterinária
12.
J Immunoassay Immunochem ; 41(2): 195-207, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31885329

RESUMO

A radioimmunoassay (RIA) method for progesterone has been developed. It has been validated for the determination of progesterone in cow skim milk. The main reagents used in the development work were prepared and characterized in-house. The assay uses a radioiodinated tracer purified by gel filtration on Sephadex G-25, standards prepared in skim milk and coated tubes with specific antibodies as the solid phase (separation system). The radiochemical purity of the tracer was greater than 95%, the maximum binding using solid phase reached 43% and the nonspecific binding didn't exceed 5%. Series of progesterone standards using milk matrix with concentration ranging from 0 to 40ng/mL were prepared. Detection limit of the assay was 0.13ng/mL and the precision evaluation gives an intra and inter-assay coefficient variations between 4.94% and 12.66%. The recovery obtained with skim milk samples was 84% to 119% and the parallelism test indicated good linearity (R2 > 0.99). The clinical tests give high correlation coefficient (r = 0.998) between progesterone concentrations of cow's skim milk assayed using developed progesterone RIA kit and commercial progesterone RIA kit.


Assuntos
Leite/química , Progesterona/análise , Radioimunoensaio , Animais , Bovinos , Progesterona/imunologia , Controle de Qualidade
13.
Reprod Biol Endocrinol ; 17(1): 113, 2019 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-31883523

RESUMO

BACKGROUND: The Hippo pathway plays critical roles in regulating cell proliferation, differentiation and survival among species. Hippo pathway proteins are expressed in the ovary and are involved in ovarian function. Deletion of Lats1 causes germ cell loss, ovarian stromal tumors and reduced fertility. Ovarian fragmentation induces nuclear YAP1 accumulation and increased follicular development. At ovulation, follicular cells stop proliferating and terminally differentiate, but the mechanisms controlling this transition are not completely known. Here we explore the role of Hippo signaling in mouse granulosa cells before and during ovulation. METHODS: To assess the effect of oocytes on Hippo transcripts in cumulus cells, cumulus granulosa cells were cultured with oocytes and cumulus oocyte complexes (COCs) were cultured with a pSMAD2/3 inhibitor. Secondly, to evaluate the criticality of YAP1 on granulosa cell proliferation, mural granulosa cells were cultured with oocytes, YAP1-TEAD inhibitor verteporfin or both, followed by cell viability assay. Next, COCs were cultured with verteporfin to reveal its role during cumulus expansion. Media progesterone levels were measured using ELISA assay and Hippo transcripts and expansion signatures from COCs were assessed. Lastly, the effects of ovulatory signals (EGF in vitro and hCG in vivo) on Hippo protein levels and phosphorylation were examined. Throughout, transcripts were quantified by qRT-PCR and proteins were quantified by immunoblotting. Data were analyzed by student's t-test or one-way ANOVA followed by Tukey's post-hoc test or Dunnett's post-hoc test. RESULTS: Our data show that before ovulation oocytes inhibit expression of Hippo transcripts and promote granulosa cell survival likely through YAP1. Moreover, the YAP1 inhibitor verteporfin, triggers premature differentiation as indicated by upregulation of expansion transcripts and increased progesterone production from COCs in vitro. In vivo, ovulatory signals cause an increase in abundance of Hippo transcripts and stimulate Hippo pathway activity as indicated by increased phosphorylation of the Hippo targets YAP1 and WWTR1 in the ovary. In vitro, EGF causes a transient increase in YAP1 phosphorylation followed by decreased YAP1 protein with only modest effects on WWTR1 in COCs. CONCLUSIONS: Our results support a YAP1-mediated mechanism that controls cell survival and differentiation of granulosa cells during ovulation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Proteínas de Ciclo Celular/fisiologia , Células da Granulosa/fisiologia , Ovulação/fisiologia , Transdução de Sinais/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Animais , Proteínas de Ciclo Celular/antagonistas & inibidores , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Proliferação de Células , Sobrevivência Celular/fisiologia , Células Cultivadas , Meios de Cultivo Condicionados/química , Células do Cúmulo/fisiologia , Fator de Crescimento Epidérmico/farmacologia , Feminino , Camundongos , Oócitos/fisiologia , Progesterona/análise , RNA Mensageiro/análise , Transdução de Sinais/genética , Verteporfina
14.
Reprod Biol Endocrinol ; 17(1): 111, 2019 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-31878927

RESUMO

BACKGROUND: Previous studies of expression profiles of major endometrial effectors of steroid physiology in endometriosis have yielded markedly conflicting conclusions, presumably because the relative effects of type of endometriosis, fertility history and menstrual cycle phases on the measured variables were not considered. In the present study, endometrial mRNA and protein levels of several effectors of steroid biosynthesis and action in patients with stage III-IV ovarian endometriosis (OE) with known fertility and menstrual cycle histories were compared with the levels in control endometrium to test this concept. METHODS: Endometrial samples were collected from patients without endometriosis (n = 32) or OE stages III-IV (n = 52) with known fertility and cycle histories. qRT-PCR and immunoblotting experiments were performed to measure levels of NR5A1, STAR, CYP19A1, HSD17Bs, ESRs and PGR transcripts and proteins, respectively. Tissue concentrations of steroids (P4, T, E1 and E2) were measured using ELISAs. RESULTS: The levels of expression of aromatase and ERß were lower (P < 0.0001) and 17ß-HSD1 (P < 0.0001) and PRA (P < 0.01) were higher in OE endometrium. Lower aromatase levels and higher 17ß-HSD1 levels were detected in fertile (aromatase: P < 0.05; 17ß-HSD1: P < 0.0001) and infertile (aromatase: P < 0.0001; 17ß-HSD1: P < 0.0001) OE endometrium than in the matched control tissues. Both proliferative (PP) and secretory (SP) phase OE samples expressed aromatase (P < 0.0001) and ERß (PP: P < 0.001; SP: P < 0.01) at lower levels and 17ß-HSD1 (P < 0.0001) and PRA (PP: P < 0.01; SP: P < 0.0001) at higher levels than matched controls. Higher 17ß-HSD1 (P < 0.01) and E2 (P < 0.05) levels and a lower (P < 0.01) PRB/PRA ratio was observed in infertile secretory phase OE endometrium than in control. CONCLUSIONS: We report that dysregulated expression of 17ß-HSD1 and PGR resulting in hyperestrogenism and progesterone resistance during the secretory phase of the menstrual cycle, rather than an anomaly in aromatase expression, was the hallmark of eutopic endometrium from infertile OE patients. Furthermore, the results provide proof of concept that the fertility and menstrual cycle histories exerted relatively different effects on steroid physiology in the endometrium from OE patients compared with the control subjects.


Assuntos
Endometriose/metabolismo , Endométrio/metabolismo , Hormônios Esteroides Gonadais/metabolismo , Doenças Ovarianas/metabolismo , Receptores de Esteroides/metabolismo , 17-Hidroxiesteroide Desidrogenases/análise , 17-Hidroxiesteroide Desidrogenases/genética , Adolescente , Adulto , Aromatase/análise , Aromatase/genética , Endométrio/química , Estradiol/análise , Feminino , Expressão Gênica , Humanos , Infertilidade Feminina/metabolismo , Ciclo Menstrual , Progesterona/análise , Receptores Estrogênicos/análise , Receptores de Progesterona/análise , Receptores de Esteroides/genética , Adulto Jovem
15.
PLoS One ; 14(10): e0224081, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31622417

RESUMO

Steroid hormones and their respective nuclear receptors are essential mediators in numerous physiologic and pathophysiologic processes, ranging from regulation of metabolism, immune function, and reproductive processes to the development of hormone-dependent cancers such as those of the breast and prostate. Because steroids must enter cells before activating nuclear receptors, understanding the mechanisms by which cellular uptake occurs is critical, yet a clear understanding of these mechanisms has been elusive. It is generally assumed that diffusion-driven uptake is similar across various steroids whereas an elevated cellular concentration is thought to reflect active uptake, but these assumptions have not been directly tested. Here we show that intact cells rapidly accumulate free steroids to markedly elevated concentrations. This effect varies widely depending on steroid structure; more lipophilic steroids reach more elevated concentrations. Strong preferences exist for 3ß-OH, Δ5-steroids vs. 3-keto, Δ4-structural features and for progestogens vs. androgens. Surprisingly, steroid-structure-specific preferences do not require cell viability, implying a passive mechanism, and occur across cells derived from multiple tissue types. Physiologic relevance is suggested by structure-specific preferences in human prostate tissue compared with serum. On the other hand, the presence of serum proteins in vitro blocks much, but not all, of the passive accumulation, while still permitting a substantial amount of active accumulation for certain steroids. Our findings suggest that both passive and active uptake mechanisms make important contributions to the cellular steroid uptake process. The role of passive, lipophilicity-driven accumulation has previously been largely unappreciated, and its existence provides important context to studies on steroid transport and action both in vitro and in vivo.


Assuntos
Esteroides/metabolismo , Androgênios/análise , Androgênios/sangue , Androgênios/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Desidroepiandrosterona/análise , Desidroepiandrosterona/metabolismo , Humanos , Cinética , Pregnenolona/análise , Pregnenolona/metabolismo , Progesterona/análise , Progesterona/metabolismo , Progesterona/farmacologia , Esteroides/análise , Esteroides/farmacologia , Espectrometria de Massas em Tandem
16.
J Dairy Sci ; 102(12): 11207-11216, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31606211

RESUMO

Evaluating fertility traits based on endocrine progesterone profiles is becoming a promising option to improve dairy cow fertility. Several studies have been conducted on endocrine fertility traits, mainly in the Holstein breed. In this study, focusing also on the Swedish Red (SR) breed, genetic parameters were estimated for classical and endocrine fertility traits, the latter based on in-line milk progesterone records obtained for 14 Swedish herds using DeLaval Herd Navigator (DeLaval International, Tumba, Sweden). A total of 210,403 observations from 3,437 lactations of 1,107 SR and 1,538 Holstein cows were used. Mixed linear animal models were used for estimation of genetic parameters. Least squares means analysis showed that Holstein cows had a 2.5-d-shorter interval from calving to commencement of luteal activity (C-LA) and longer length of first inter-ovulatory interval (IOI) than SR cows. The highest mean interval for C-LA, IOI, and first luteal phase length (LPL) was observed in the fourth parity. The incidence of short (<18 d), normal, (18-24 d), and long (>24 d) IOI was 29.3, 40.7, and 30%, respectively. Genetic analysis indicated moderate heritability (h2) for C-LA (h2 = 0.24), luteal activity during the first 60 d in milk (LA60, h2 = 0.15), proportion of samples with luteal activity (PLA, h2 = 0.13), and calving to first heat (CFH, h2 = 0.18), and low heritability estimates for LPL (h2 = 0.08) and IOI (h2 = 0.03) in the combined data set for both breeds. Similar heritability estimates were obtained for each breed separately except for IOI and LPL in SR cows, for which heritability was estimated to be zero. Swedish Red cows had 0.01 to 0.06 higher heritability estimates for C-LA, LA60, and PLA than did Holstein cows. Calving interval had moderate heritability among the classical traits for Holstein and the combined data set, but h2 was zero for SR. Commencement of luteal activity had a strong genetic correlation with LA60 (mean ± SE; -0.88 ± 0.06), PLA (-0.72 ± 0.11), and CFH (0.90 ± 0.04). Similarly, CFH had a strong genetic correlation with IOI (0.98 ± 0.20). Number of inseminations per series showed a weak genetic correlation with all endocrine traits except IOI. Overall, endocrine traits had higher heritability estimates than classical traits in both breeds, and may have a better potential to explain the actual reproductive status of dairy cows than classical traits. This might favor inclusion of some endocrine fertility traits-especially those related to commencement of luteal activity-as selection criteria and breeding goal traits if recording becomes more common in herds. Further studies on genetic and genomic evaluations for endocrine fertility traits may help to provide firm conclusions. A prerequisite is that the data from automatic devices be made available to recording and breeding organizations in the future and included in a central database.


Assuntos
Bovinos/genética , Fertilidade/genética , Leite/química , Progesterona/análise , Animais , Feminino , Testes Genéticos/veterinária , Lactação , Análise dos Mínimos Quadrados , Modelos Lineares , Gravidez , Suécia
17.
J Dairy Sci ; 102(12): 11104-11115, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31606217

RESUMO

Our aim was to estimate genetic parameters of atypical reproductive patterns and estimate their genetic correlation with milk production and classical fertility traits for commercial dairy cows. In contrast with classical fertility traits, atypical reproductive patterns based on in-line milk progesterone profiles might have higher heritability and lower genetic correlation with milk production. We had in-line milk progesterone profiles available for 12,046 cycles in 4,170 lactations of 2,589 primiparous and multiparous cows (mainly Holstein Friesian) from 14 herds. Based on progesterone profiles, 5 types of atypical reproductive patterns in a lactation were defined: delayed ovulation types I and II, persistent corpus luteum types I and II, and late embryo mortality. These atypical patterns were detected in 14% (persistent corpus luteum type II) to 21% (persistent corpus luteum type I) of lactations. In 47% of lactations, at least 1 atypical pattern was detected. Threshold model heritabilities for atypical reproduction patterns ranged between 0.03 and 0.14 and for most traits were slightly higher compared with classical fertility traits. The genetic correlation between milk yield and calving interval was 0.56, whereas genetic correlations between milk yield and atypical reproductive patterns ranged between -0.02 and 0.33. Although most of these correlations between milk yield and atypical reproductive patterns are still unfavorable, they are lower compared with the correlations between classical fertility traits and milk yield. Therefore selection against atypical reproductive patterns may relax some constraints in current dairy breeding programs, to enhance genetic progress in both fertility and milk yield at a steady pace. However, as long as the target trait for fertility is calving interval, atypical reproductive patterns will not add additional value to the breeding goal in the near future due to the low number of available records.


Assuntos
Bovinos/genética , Leite/química , Progesterona/análise , Reprodução/genética , Animais , Cruzamento , Bovinos/fisiologia , Corpo Lúteo , Feminino , Fertilidade/genética , Lactação/genética , Paridade , Fenótipo , Gravidez
18.
Zhongguo Zhong Yao Za Zhi ; 44(15): 3239-3245, 2019 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-31602878

RESUMO

The present study was conducted to explores the effects of short-term addition of 17ß-E2 on the growth,gonad development and internal quality of overwintering Whitmania pigra. Before overwintering,0. 0,1. 0,10. 0,25. 0,50. 0,100. 0 µg·L~(-1) of 17ß-E2 were added to the aquaculture water for 6 weeks and then hibernated for 60 days. The changes of growth performance,gonad index,morphological structure of spermary( ovary),endogenous steroid hormones level and internal quality were measured. The results showed that the body weight,weight gain rate,specific growth rate,female gonad index,oocyte development and endogenous estrogen level of the leech increased first and then decreased with the increase of the concentration of exogenous 17ß-E2,which were higher than those of the control group. The body weight,weight gain rate and specific growth rate of the leech at the concentration of 25 µg·L~(-1)17ß-E2 were significantly higher than those of the other groups( P<0. 05),oocyte development and endogenous estrogen levels were significantly higher than those of other groups at the concentration of 50 µg·L~(-1)( P<0. 05). When the concentration of exogenous 17ß-E2 was higher than 50 µg·L~(-1),the levels of male gonad index,spermatocyte development,endogenous androgen and progesterone were significantly inhibited( P< 0. 05). There was no significant difference in endogenous corticosteroid levels among the groups. In conclusion,short-term addition of exogenous 17ß-E2 of 10-25 µg·L~(-1) could promote the growth of overwintering leeches,oocyte development and antithrombin activity without inhibiting the development of male gonads.


Assuntos
Estradiol/farmacologia , Gônadas/crescimento & desenvolvimento , Sanguessugas/efeitos dos fármacos , Sanguessugas/crescimento & desenvolvimento , Androgênios/análise , Animais , Estrogênios/análise , Feminino , Gônadas/efeitos dos fármacos , Hibernação , Masculino , Progesterona/análise
19.
Anal Biochem ; 587: 113461, 2019 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-31577917

RESUMO

There is a pressing need for simple and accurate analytical tools to assess the level of EDCs in environmental samples. In this work, a simple and highly sensitive competitive lateral flow assay (LFA) was developed for progesterone (P4). Gold nanoparticles (AuNPs) were functionalized with a previously isolated 60-mer aptamer for P4 and further hybridized with 8-mer complementary sequence modified with biotin. In the absence of P4, AuNP-duplexed aptamer conjugates are capture by a test line made with streptavidin. Conformational change within aptamer sequence upon target recognition causes the release of the biotinylated complementary sequence and disappearance of the colored test line. By optimizing the hybridization location of the 8-mer biotinylated sequences, the sensitivity of sensor was improved by 20-folds to achieve 5 nM detection level of P4 in buffer and spiked tap water samples. The simply fabricated sensor demonstrated a dynamic range in the lower nanomolar range and excellent selectivity against potential interfering molecules including the closely similar 17ß-estradiol (E2). The sensor can be used as a fast screening tool to assess the level of P4 in water. Implementing the developed assay avoids applying laborious extraction procedures and lengthily analysis by conventional chromatography based instruments.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais , Progesterona/análise , Ouro/química , Nanopartículas Metálicas/química
20.
Placenta ; 87: 30-37, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31542634

RESUMO

BACKGROUND: Human syncytiotrophoblast mitochondria require the activity of the isocitrate dehydrogenase type 2 (IDH2) to obtain reduced coenzymes for progesterone (P4) synthesis. Data from the literature indicate that mitochondrial steroidogenic contact sites transform efficiently cholesterol into P4. In this research, we identified the IDH2 as a member of the steroidogenic contact site and analyzed the steroidogenic role of its activity. METHOD: Human syncytiotrophoblast mitochondria were isolated by differential centrifugation, and steroidogenic contact sites were obtained by osmotic shock and sucrose gradient ultracentrifugation. In-gel native activity assay, mass spectroscopy, and western blot were used to identify the association of proteins and their activities. P4 was determined by immunofluorescence. RESULTS: The IDH2 was mainly identified in steroidogenic contact sites, and its activity was associated with a complex of proteins with an apparent molecular mass of ~590 kDa. Mass spectroscopy showed many groups of proteins with several metabolic functions, including steroidogenesis and ATP synthesis. The IDH2 activity was coupled to P4 synthesis since in the presence of Ca2+ or Na2SeO3, inhibitors of the IDH2, the P4 production decreased. CONCLUSIONS: The human syncytiotrophoblast mitochondria build contact sites for steroidogenesis. The IDH2, a non-membrane protein, supplies the NADPH required for the synthesis of P4 in a complex (steroidosome) that associate the proteins required to transform efficiently cholesterol into P4, which is necessary in pregnancy to maintain the relationship between mother and fetus. GENERAL SIGNIFICANCE: The IDH2 is proposed as a check point in the regulation of placental steroidogenesis.


Assuntos
Isocitrato Desidrogenase/metabolismo , Complexos Multiproteicos/metabolismo , Placenta/metabolismo , Progesterona/metabolismo , Esteroides/biossíntese , Adolescente , Adulto , Feminino , Humanos , Mitocôndrias/química , Mitocôndrias/metabolismo , Gravidez , Progesterona/análise , Ligação Proteica , Esteroides/análise , Trofoblastos/química , Trofoblastos/metabolismo , Trofoblastos/ultraestrutura , Adulto Jovem
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