Germline ATM Mutations Detected by Somatic DNA Sequencing in Lethal Prostate Cancer

DNA damage response; PARP inhibition; Prostate cancerRespuesta al daño del ADN; Inhibición de PARP; Cáncer de próstataResposta al dany de l'ADN; Inhibició de PARP; Càncer de pròstataBackground Germline mutations in the ataxia telangiectasia mutated (ATM) gene occur in 0.5–1% of the overall population and are associated with tumour predisposition. The clinical and pathological features of ATM-mutated prostate cancer (PC) are poorly defined but have been associated with lethal PC. Objective To report on the clinical characteristics including family history and clinical outcomes of a cohort of patients with advanced metastatic castration-resistant PC (CRPC) who were found to have germline ATM mutations after mutation detection by initial tumour DNA sequencing. Design, setting, and participants We acquired germline ATM mutation data by saliva next-generation sequencing from patients with ATM mutations in PC biopsies sequenced between January 2014 and January 2022. Demographics, family history, and clinical data were collected retrospectively. Outcome measurements and statistical analysis Outcome endpoints were based on overall survival (OS) and time from diagnosis to CRPC. Data were analysed using R version 3.6.2 (R Foundation for Statistical Computing, Vienna, Austria). Results and limitations Overall, seven patients (n = 7/1217; 0.6%) had germline ATM mutations detected, with five of them having a family history of malignancies, including breast, prostate, pancreas, and gastric cancer; leukaemia; and lymphoma. Two patients had concomitant somatic mutations in tumour biopsies in genes other than ATM, while two patients were found to carry more than one ATM pathogenic mutation. Five tumours in germline ATM variant carriers had loss of ATM by immunohistochemistry. The median OS from diagnosis was 7.1 yr (range 2.9–14 yr) and the median OS from CRPC was 5.3 yr (range 2.2–7.3 yr). When comparing these data with PC patients sequenced by The Cancer Genome Atlas, we found that the spatial localisation of mutations was similar, with distribution of alterations occurring on similar positions in the ATM gene. Interestingly, these include a mutation within the FRAP-ATM-TRRAP (FAT) domain, suggesting that this represents a mutational hotspot for ATM. Conclusions Germline ATM mutations are rare in patients with lethal PC but occur at mutational hotspots; further research is warranted to better characterise the family histories of these men and PC clinical course

Caracterização do estro de novilhas cruzadas (Bos taurus indicus x Bos taurus taurus) por radiotelemetria

Although artificial insemination (AI) has countless advantages, in Brazil this technique is performed in only 7% of beef cattle. Failures on estrous detection are the main limiting factor to the success of this technique. An electronical system, based on radiotelemetry, was developed for estrous detection. This system provides day, hour and service length. The present study aimed to evaluate by radiotelemetry system the behavioral characteristics of the estrous of crossbred heifers (Bos taurus taurus x Bos taurus indicus) raised at pasture regimen in southwest region of Brazil. The hypothesis is that estrous lenght and service frequency are variable among females and that most of services occur at nocturnal period. For estrous synchronization, heifers received 0,5mg of MGA/animal/day, once a day during 8 days, and 15mg of luprostiol (PG), IM, at the last day of MGA treatment. Estrous were registered in a radiotelemetry system, denominated “Heat-Watch”, during a 120 hours period after PG injection. The mean of estrous length was 10,4 + 5,7 hours (ranging from 45 minutes to 22,7 hours). The mean of number of services was 26,2 + 13,6 (ranging from 3 to 81 services). The mean of services length was 2,7 + 0,3 seconds. Dransfield et al.4 classified the estrous in short (< 7 hours) and long (>; 7 hours) length and low (< 1,5 services/hour) and high (>; 1,5 services/hour) intensity. There was a higher incidence of long length when compared to short lenght (72,8% vs. 27,2%; P<0,05) and higher incidence of high intensity estrous when compared to that of low intensity (70,2% vs. 29,8%; P<0,05). The mean of services at day period (7:00 AM to 7:00 PM) was 10,0 + 9,7 and at night period was 13,0 + 12,4 (7:00 PM to 7:00 AM). Effect of period on services number was not observed (P=0,08). In the present study it was confirmed the hypothesis that estrous behavior is extremely variable among females, however, the hypothesis that most services occur at night period was not confirmed.Embora a técnica de inseminação artificial (IA) apresente inúmeras vantagens, no Brasil é empregada em apenas 7% das fêmeas de corte. Falhas na detecção de estros constituem o principal fator limitante para a obtenção de êxito no emprego da técnica. Um sistema eletrônico, que se baseia na radiotelemetria, foi desenvolvido para a detecção de estros. Este sistema fornece o registro da data, horário e duração das montas ocorridas. O presente estudo teve como objetivo avaliar, pelo sistema de radiotelemetria, as características comportamentais de estro em novilhas cruzadas de corte (Bos taurus taurus x Bos taurus indicus), criadas em regime extensivo, na região sudoeste do Brasil. A hipótese testada foi que a duração dos estros e o número de montas são bastante variáveis entre as fêmeas e que a maioria das montas ocorre no período noturno. Para a sincronização dos estros as novilhas receberam 0,5mg de MGA/cabeça/dia, uma vez ao dia, durante 8 dias, e uma injeção de 15mg de luprostiol (PG) via IM no último dia da ingestão de MGA. Os estros foram registrados pelo sistema de radiotelemetria "Heat-Watch", durante um período de até 120 horas após a aplicação de PG. A duração média dos estros foi de 10,4 + 5,7 horas, duração que variou de 45 minutos a 22,7 horas. O número médio de montas foi de 26,2 + 13,6 e variou de 3 a 81 montas. A duração média das montas foi de 2,7 + 0,3 segundos. Dransfield et al.¹ classificaram os estros em curta (< 7 horas) e longa (>; 7 horas) duração e baixa (< 1,5 montas/hora) e alta (>; 1,5 montas/hora) intensidade. Houve uma maior incidência dos estros de longa duração quando comparados aos de curta duração (72,8% vs. 27,2%; P<0,05) e uma maior incidência dos estros de alta intensidade quando comparados aos de baixa intensidade (70,2% vs. 29,8%; P<0,05). A média geral de montas diurnas foi de 10,0 + 9,7 (das 7:00 às 19:00 horas) e de montas noturnas de 13,0 + 12,4 (das 19:00 às 7:00 horas), sendo que não foi observado efeito de período do dia no número de montas (P=0,08). No presente estudo confirmou-se a hipótese de que o comportamento de estro é extremamente variável entre as fêmeas, mas não a de que a maioria das montas ocorre no período noturno

Elucidating Prostate Cancer Behaviour During Treatment via Low-pass Whole-genome Sequencing of Circulating Tumour DNA

Cabazitaxel; Cell-free DNA; Tumour fractionCabazitaxel; ADN lliure de cèl·lules; Fracció tumoralCabazitaxel; ADN libre de células; Fracción tumoralBackground Better blood tests to elucidate the behaviour of metastatic castration-resistant prostate cancer (mCRPC) are urgently needed to drive therapeutic decisions. Plasma cell-free DNA (cfDNA) comprises normal and circulating tumour DNA (ctDNA). Low-pass whole-genome sequencing (lpWGS) of ctDNA can provide information on mCRPC behaviour. Objective To validate and clinically qualify plasma lpWGS for mCRPC. Design, setting, and participants Plasma lpWGS data were obtained for mCRPC patients consenting to optional substudies of two prospective phase 3 trials (FIRSTANA and PROSELICA). In FIRSTANA, chemotherapy-naïve patients were randomised to treatment with docetaxel (75 mg/m2) or cabazitaxel (20 or 25 mg/m2). In PROSELICA, patients previously treated with docetaxel were randomised to 20 or 25 mg/m2 cabazitaxel. lpWGS data were generated from 540 samples from 188 mCRPC patients acquired at four different time points (screening, cycle 1, cycle 4, and end of study). Outcome measurements and statistical analysis lpWGS data for ctDNA were evaluated for prognostic, response, and tumour genomic measures. Associations with response and survival data were determined for tumour fraction. Genomic biomarkers including large-scale transition (LST) scores were explored in the context of prior treatments. Results and limitations Plasma tumour fraction was prognostic for overall survival in univariable and stratified multivariable analyses (hazard ratio 1.75, 95% confidence interval 1.08–2.85; p = 0.024) and offered added value compared to existing biomarkers (C index 0.722 vs 0.709; p = 0.021). Longitudinal changes were associated with drug response. PROSELICA samples were enriched for LSTs (p = 0.029) indicating genomic instability, and this enrichment was associated with prior abiraterone and enzalutamide treatment but not taxane or radiation therapy. Higher LSTs were correlated with losses of RB1/RNASEH2B, independent of BRCA2 loss. Conclusions Plasma lpWGS of ctDNA describes CRPC behaviour, providing prognostic and response data of clinical relevance. The added prognostic value of the ctDNA fraction over established biomarkers should be studied further.This work was supported by Prostate Cancer UK and the Movember Foundation through the London Movember Centre of Excellence (CEO13-2-002), Cancer Research UK (Centre Programme grant), Experimental Cancer Medicine Centre grant funding from Cancer Research UK and the Department of Health, and Biomedical Research Centre funding to the Royal Marsden ECMC (CRM064X). George Seed was funded by a Prostate Cancer UK PhD Studentship (grant ref. TLD-S15-006, 2016–2020) and a Prostate Cancer UK research fellowship (grant ref. TLD-PF19-005, from 2021). Semini Sumanasuriya was funded by a Prostate Cancer UK grant (ref. RIA1 5-ST2-O18). The authors are grateful for support and funding from Sanofi Aventis. The sponsor played a role in collection of the data and review and approval of the manuscript

Endometrial prostaglandin F2α in vitro production and its modulation regarding dominant follicle position in cattle

Prostaglandin F2α (PGF2α) determines luteolysis in cattle, and the ability to manipulate its endogenous synthesis is indispensible for large-scale animal breeding. Estradiol (E2) and progesterone (P4) modulate several molecular pathways in endometrial cells, including the synthesis of PGF2α; however, its specific mechanisms are still not totally known. This study investigated the production in vitro and possible modulation of endometrial PGF2α due to a local effect of endogenous E2 in the ipsilateral uterine horn (UH) containing the dominant follicle (DF) or from P4 in ipsilateral horn containing the corpus luteum (CL). The PGF2α stimulators oxytocin (OT) and phorbol 12,13-dibutyrate (PDBu) were incubated with endometrial explants, and PGF2α content was measured. For that, cycling cows were synchronized, the development of DF and CL was examined by ultrasonography and on the seventh day of the estrous cycle, endometrial explants were collected and cultured in medium supplemented with 10-6 M PDBu or 10-6 M OT or non-supplemented. Media samples were collected immediately after treatment and 60 min later. Radioimmunoassay showed that the PGF2α content of the UH ipsilateral to the DF was 49% less than that of the contralateral UH (8.22 ± 0.95 vs. 12.24 ± 0.95 pg/mL/mg tissue, respectively; P < 0.01). However, the PGF2α levels did not differ between the UHs as a function of the CL position (9.46 ± 0.95 vs. 11 ± 0.95 pg/mL/mg; P > 0.05). The cellular stimulators promoted an increase in PGF2α synthesis (P < 0.02), and the effects differed among the animals (P < 0.04). The PGF2a production was higher in the explants treated with PDBu rather than OT (13.68 ± 1.16 vs. 10.01 ± 1.16 pg/mL/mg tissue, respectively; P < 0.05). In conclusion, PGF2α synthesis is modulated by the presence of the DF (local E2) but not the CL (local P4), and both PDBu and OT stimulated PGF2a synthesis.A prostaglandina F2α (PGF2α) determina a luteólise em bovinos. A capacidade de manipular sua síntese endógena é indispensável para a produção animal em grande escala. O estradiol (E2) e a progesterona (P4) modulam diversas vias moleculares das células endometriais, incluindo a síntese de PGF2α; no entanto, pouco se sabe sobre seus mecanismos específicos. Este trabalho investigou a produção in vitro e a possível modulação da PGF2α endometrial devido a um efeito local do E2 endógeno no corno uterino ipsilateral ao folículo dominante (FD) ou da P4 no corno ipsilateral ao corpo lúteo (CL). Os estimuladores de PGF2α oxitocina (OT) e 12,23-dibutirato de forbol (PDBu) foram incubados com explantes endometriais, e o conteúdo de PGF2α foi mensurado. Para tal, vacas cíclicas foram sincronizadas, o desenvolvimento de FD e CL foi examinado por ultrassonografia, e no 17º dia do ciclo estral os explantes endometriais foram coletados e cultivados em meio ou suplementados com PDBu 10-6M ou 10-6M OT. As amostras de meio foram coletadas imediatamente após o tratamento e sessenta minutos depois. O radioimunoensaio mostrou que o conteúdo de PGF2α do corno ipsilateral ao FD foi 49% menor que o do corno contralateral (8,22 ± 0,95 vs. 12,24 ± 0,95 pg/mL/mg de tecido, respectivamente, P < 0,01). No entanto, os níveis de PGF2α não diferiram entre os cornos em função da posição do CL (9,46 ± 0,95 versus 11 ± 0,95 pg/mL/mg; P > 0,05). Os estimuladores celulares promoveram um aumento na síntese de PGF2α (P < 0,02), e os efeitos diferiram entre os animais (P < 0,04). A produção de PGF2α foi maior nos explantes tratados com PDBu em comparação à OT (13,68 ± 1,16 versus 10,01 ± 1,16 pg/mL/mg de tecido, respectivamente, P < 0,05). A conclusão obtida foi que a síntese de PGF2α é: modulada pela presença do FD (E2 local), mas não do CL (P4 local); e estimulada por PDBu e OT

Mecanismos endócrinos e moleculares envolvidos na formação do corpo lúteo e na luteólise: revisão de literatura

The corpus luteum (CL) is a transitory endocrinal structure formed by steroidogenic small luteal cells (SLC) and large luteal cells (LLC) that associated with fibroblast and a wide web of capillaries form a structure specialized in synthesis of progesterone (P4). In general, for the synthesis of P4 in the steroidogenic luteal cells, cholesterol joints specific receptors on the cellular membrane and is transported to the cytosol. Later, cholesterol goes to an internal mitochondrial membrane and by the action of the enzyme P450scc is transformed into pregnenolone. In the smooth endoplasmic reticulum pregnenolone is converted to P4 by the enzyme 3²-hydroxysteroid dehydrogenase (3²-HSD). The steroidogenic acute regulatory protein (StAR), the peripheral benzodiazepine receptor (PBR) and endozepine participate in the transport of cholesterol to the different mitochondrial compartments. Therefore, it is supposed that the capacity of synthesis of P4 in the CL is related to the cellular concentration of receptors that catch cholesterol, to the enzymes P450scc and 3²-HSD and to the cholesterol cellular transport proteins. In bovine, the LLC are responsible for more than 80% of P4 production in the CL. The lowest concentration of cholesterol transport proteins in the mitochondria seems to limit the synthesis of P4 in the SLC. P4 supports a proper uterine environment for the development of conceptuses, depending on the specie. In most species, the lack of fertilization or the conceptus incapacity to signalize its presence in the uterus establishes luteolysis. This physiological event is characterized by the functional and structural regression of the CL. For the establishment and maintenance of pregnancy it is necessary to block luteolysis through different mechanisms among species. In primates and equids it occurs by the secretion of specific gonadotropins and in ruminants by antiluteolytic factors. This review has the objective to characterize the endocrine and molecular mechanisms involved in the formation of the corpus luteum and luteolysis.O corpo lúteo (CL) é uma estrutura endócrina transitória formada por células luteais esteroidogênicas pequenas (SLC) e grandes (LLC), que associadas aos fibroblastos e a uma ampla rede de capilares constituem uma estrutura especializada na síntese de progesterona (P4). De maneira geral, para a síntese de P4 nas células luteais esteroidogênicas, o colesterol se liga a receptores específicos na membrana celular e é transportado ao citosol. Posteriormente, o colesterol dirige-se a membrana mitocondrial interna e por ação da enzima P450scc transforma-se em pregnenolona. No retículo endoplasmático liso a pregnenolona é convertida a P4 pela enzima 3²-hidroxiesteróide deidrogenase (3²-HSD). A proteína de regulação aguda da esteroidogênese (StAR), o receptor benzodiazepínico tipo periférico (PBR) e a endozepina participam no transporte do colesterol para os diferentes compartimentos mitocondriais. Assim, supõe-se que a capacidade de síntese de P4 no CL esteja relacionada à concentração celular de receptores que captam o colesterol, às enzimas P450scc e 3²-HSD, e às proteínas celulares transportadoras de colesterol. Na espécie bovina, as LLC são responsáveis por mais de 80% da produção deste hormônio no CL. A menor concentração de proteínas transportadoras de colesterol na mitocôndria parecem limitar a síntese de P4 nas SLC. A P4 favorece um meio ambiente uterino apropriado para o desenvolvimento do(s) concepto(s), dependendo da espécie. Na maioria das espécies, a ausência da fertilização ou a incapacidade do concepto em sinalizar sua existência no útero estabelecem a luteólise. Tal evento fisiológico caracteriza-se pela regressão funcional e estrutural do(s) corpo(s) lúteo(s). Para o estabelecimento e a manutenção da prenhez torna-se necessário o bloqueio da luteólise por mecanismos que diferem entre as espécies. Em primatas e equídeos esse reconhecimento ocorre pela secreção de gonadotrofinas específicas e em ruminantes pela presença de fatores anti-luteolíticos. Esta revisão tem como objetivo caracterizar os mecanisnos endócrinos e moleculares envolvidos na formação do corpo lúteo e na luteólise

Use of protein kinase C and phospholipase A2 inhibitors in bovine endometrial cells treated with estradiol and calcium ionophore

The release of endometrial prostaglandin-F2α (PGF2α) in bovine females can be induced in vivo by estradiol (E2). However, its role in this mechanism has not been clarified. We hypothesized that E2 stimulates the activity and abundance of protein kinase C (PKC) and phospholipase A2 (PLA2). Our objective in this study was to analyze the effects of PKC and PLA2 inhibitors on PGF2α synthesis induced by E2 and calcium ionophore (CI) in bovine endometrial cells (BEND cells; Experiment 1). Additionally, we evaluated the abundance of PKC and PLA2 in endometrial explants of cows treated or not with E2 17 days after estrus (D17, D0 = estrus; Experiment 2). In Experiment 1, BEND cells were submitted to a PKC inhibitor (10 μM of C25H24N4O2; bisindolylmaleimide I, or BIS I), a PLA2 inhibitor (20 μM of arachydoniltrifluoromethane or AACOCF3), or none. The BEND cells were subsequently treated with E2 and CI, and PGF2α concentrations were measured in the culture medium through radioimmunoassay. For DIF-12 (PGF2α concentration 12 h after treatment subtracted from PGF2α concentration at hour 0), no PKC inhibitor effect was observed (P= 0.2709). However, DIF-12 was lower (P < 0.05) for groups treated with the PLA2 inhibitor and PLA2 inhibitor + CI + E2 groups than the control and CI + E2 groups. Thus, AACOCF3 was an efficient PLA2 inhibitor in the BEND cells culture system, and E2 did not stimulate the synthesis of PKC and PLA2. In Experiment 2, cyclic Nellore heifers received none (n = 5) or 3 mg (n = 6) of 17β-E2 on D17 and were slaughtered 2 h after administration. The abundance of PKC and PLA2 in the endometrial tissue was evaluated using Western blotting analysis. No E2 effect was observed on PKC (P = 0.08) and PLA2 (P = 0.56). We concluded that E2 did not stimulate the activity and abundance of PKC and PLA2.A liberação endometrial de prostaglandina-F2α (PGF2α) em fêmeas bovinas pode ser induzida in vivo pelo estradiol (E2). Entretanto o seu mecanismo de ação ainda não foi bem esclarecido. Nossa hipótese é que o E2 estimula a atividade e a abundância da proteína quinase C (PKC) e da fosfolipase A2 (PLA2). Nosso objetivo com este estudo foi analisar os efeitos de inibidores de PKC e PLA2 na síntese de PGF2α induzida por E2 e ionóforo de cálcio (CI) em células endometriais bovinas (células BEND; Experimento 1). Adicionalmente, nós avaliamos a abundância de PKC e PLA2 em explantes endometriais de vacas tratadas com ou sem E2 17 dias após o estro (D17, D0 = estro; Experimento 2). No Experimento 1, células BEND foram submetidas ao inibidor de PKC (10 μM de C25H24N4O2; bisindolylmaleimide I, ou BIS I), e ao inibidor de PLA2 (20 μM de arachydoniltrifluoromethane ou AACOCF3) ou a nenhum inibidor. As células BEND foram subsequentemente tratadas com E2 e CI e concentrações de PGF2α foram mensuradas no meio de cultura por radioimunoenssaio. Para DIF-12 (concentração de PGF2α 12 horas depois do tratamento, subtraída da concentração de PGF2α na hora 0), não foi observado efeito do inibidor de PKC (P = 0.2709). Entretanto DIF-12 foi menor (P < 0.05) nos grupos tratados com inibidor de PLA2 e inibidor de PLA2 + CI + E2 quando comparados com o grupo controle e o grupo CI + E2. O AACOCF3 foi um eficiente inibidor de PLA2 em sistema de cultura de células BEND e o E2 não estimulou a síntese de PKC e PLA2. No Experimento 2, novilhas Nelore cíclicas receberam 3 mg de 17β-E2 (n = 6) ou nenhum tratamento (n = 5) no D17 e foram abatidas duas horas depois da administração dos tratamentos. A quantidade de PKC e PLA2 no tecido endometrial foi avaliada pela técnica de Western Blotting. Não foi observado efeito do E2 sobre a PKC (P= 0.08) e nem sobre a PLA2 (P= 0.56). Conclui-se que o E2 não estimulou a atividade e abundância de PKC e PLA2

Controle da síntese de prostaglandina F2± no endométrio bovino in vitro

Synthesis of endometrial prostaglandin F2± (PGF2±) results from a complex cascade of highly coordinated intracellular events. Production of PGF2± can be stimulated by mellitin and 12, 13 phorbol dibutyrate (PDBu). Objective of the present study was to verify whether basal or stimulated synthesis of PGF2± was dependent on de novo protein synthesis. In Experiment 1, endometrial explants from cyclic, non-lactating cross-bred beef cows (n=2) on day 15 of the estrous cycle were incubated in quadruplicate in culture KHB culture medium or medium supplemented with 10-6M PDBu, 10-6M melittin or 10-5M melittin. Medium samples were collected immediately and 60 minutes after treatment administration and concentration of PGF2± in medium were measured by radioimmunoassay. Concentrations of PGF2± in medium were higher (P<0.06) after treatment with PDBu in comparison with control and melittin. In Experiment 2, endometrial explants from cyclic, non-lactating cross-bred beef cows (n=4) on day 17 of the estrous cycle were incubated in quadruplicate in medium supplemented with 0, 50, 100 or 200mg cyclohexamide (CHX) and 0 or 100ng/ml PDBu, in a 4 x 2 factorial arrangement. Medium samples were collected immediately and 60 minutes after administration of treatments and concentrations of PGF2± measured by radioimmunoassay. Endometrial integrity was was evaluated by histology. Treatment with PDBu stimulated production of PGF2± (P<0.01), regardless of concentration of CHX used. The CHX did not affect production of PGF2±, either in the presence or absence of PDBu. Histological integrity of explants was preserved. It was concluded that both basal and PDBu-stimulated PGF2± production are not dependent on new protein sythesis.A síntese de prostaglandina F2± (PGF2±) endometrial, resulta de uma complexa cascata de eventos intracelulares que ocorrem de maneira altamente coordenada. A síntese de PGF2± pode ser estimulada na presença da melitina e do forbol 12,13 dibutirato (PDBu). O objetivo do presente estudo foi verificar se a produção basal e a estimulação aguda de PGF2± são dependentes da síntese de proteínas. No experimento 1, explantes obtidos de fêmeas bovinas (n=2), cíclicas, não lactantes, no dia 15 do ciclo estral foram incubados em quadruplicata, com meio de cultivo (KHB) ou KHB suplementado com PDBu 10-6M, melitina 10-6M ou melitina 10-5M. Amostras do meio foram coletadas 0 e 60 minutos após os tratamentos e as concentrações de PGF2± foram mensuradas por radioimunoensaio. Com 60 minutos após os tratamentos houve aumento das concentrações médias de PGF2± (P<0,06) em resposta ao tratamento com PDBu comparado ao grupo KHB e melitina. No experimento 2, explantes endometriais de fêmeas bovinas (n=4), não gestantes, no 17° dia do ciclo estral, pesando de 80 a 100mg foram incubados em KHB suplementado com 0, 50, 100 ou 200mg/mL de CHX e 0 ou 100ng/mL de PDBu em um arranjo fatorial 2 x 4, em quadruplicata. Amostras do meio foram coletadas 0 e 60 minutos após os tratamentos e as concentrações de PGF2± foram mensuradas por radioimunoensaio. A integridade dos explantes endometriais tratados com CHX foi avaliada por cortes histológicos. Foi observado aumento na produção de PGF2± em resposta ao tratamento com PDBu (P<0,01), entretanto, não houve diferenças na produção de PGF2± pelos tecidos tratados com diferentes concentrações de CHX, associadas ou não ao PDBu. A integridade histológica dos explantes foi preservada. Conclui-se que a produção basal e a estimulação aguda da síntese de PGF2± não são dependentes da síntese de novas proteínas

Avaliação da reutilização de implantes auriculares contendo norgestomet associados ao valerato ou ao benzoato de estradiol em vacas nelore inseminadas em tempo fixo

Em fêmeas bovinas a utilização da técnica de inseminação artificial em tempo fixo (IATF) é possibilitada pelo emprego de fármacos que tem como objetivos sincronizar a emergência das ondas foliculares, os estros e a ovulação. Em rebanhos comerciais, o custo de tais fármacos deve estabelecer uma vantajosa relação com os benefícios. O presente estudo, objetivou comparar as taxas de prenhez em vacas Nelore (Bos taurus indicus) inseminadas em tempo fixo, tratadas com implantes auriculares contendo 3mg de norgestomet (Crestar®), novos (IN) ou utilizados uma vez (IR; reutilizados), associados a administração de norgestomet (NG) e valerato de estradiol (VE) ou progesterona (P4) e benzoato de estradiol (BE). Vacas Nelore PO (n=241), amamentando, com o bezerro ao pé, receberam um dos quatro tratamentos: Crestar®novo durante 10 dias associado a administração de 3mg de NG e 5mg de VE (grupo IN/NG+VE; n=61); Crestar®novo inserido durante 8 dias associado a 50mg de P4 e 2mg de BE (grupo IN/P4+BE; n=61); Crestar®reutilizado inserido durante 10 dias associado a administração de 3mg de NG e 5mg de VE (grupo IR/NG+VE; n=58) ou Crestar®reutilizado inserido durante 8 dias associado a e 50mg de P4 e 2mg de BE (grupo IR/P4+BE; n=61). No dia da remoção dos implantes os animais receberam 7,5mg de Luprostiol e 24 horas após a remoção 1mg de BE. A IATF foi realizada 54 a 56 horas após a retirada dos implantes. Após a IATF, as vacas tiveram os estros observados durante um período de 49 dias e em estro foram reinseminadas 12 horas após a observação. O diagnóstico de gestação foi realizado por ultra-sonografia 35 dias após a IATF e após o final da estação de monta. Foram avaliadas as taxas de prenhez na IATF (TP IATF) e no final da estação de monta (TP EM). Não houve interação entre as características dos implantes (novos e reutilizados) e os tratamentos administrados no dia da inserção do implante (NG+VE e P4+BE). A utilização do CIDR®novo ou reutilizado não teve efeito nas TP IATF (48,3% vs 48,7%) e nas TP EM (85,2 vs 86,5%). Os tratamentos com NG+VE e P4+BE não tiveram efeito nas TP IATF (49,5 vs 47,5%) e TP EM (86,5 vs 85,2%). Houve efeito do número de parições (primíparas e multíparas) nas TP IATF (35% vs. 52,7%; P<0,01) e nas TP EM (71,9% vs. 90,2%; P<0,01). Conclui-se que implantes de norgestomet novos e reutilizados, quando associados ao NG+VE ou P4+BE promovem taxas de prenhez bastante satisfatórias em vacas Nelore e que melhores taxas são obtidas em fêmeas multíparas.The use of fixed-time artificial insemination (IATF) in cows is possible by the use of drugs that aim at the synchronization of the folicular wave pool, the estrus and ovulation. In trading cattle the costs of these drugs must stablish a profitable relation with the benefits. The present study meant to compare the pregnancy rates in Nelore (Bos taurus indicus) cows inseminated at fixed-time, treated with new (IN) or once used (IR; reused) auricular releasing devices with 3mg of norgestomet (Crestar®), associated with the administration of norgestomet (NG) and estradiol valerate (VE) or progesterone (P4) and estradiol benzoate (BE). Pure breed Nelore cows (n=241), on lactation with calf received one of the four treatments: new Crestar®during 10 days administrated with 3mg of NG and 5 mg of VE (group IN/NG+VE; n=61); new Crestar®inserted during 8 days with 50mg of P4 and 2mg of BE (group IN/P4+BE; n=61); reused Crestar®inserted during 10 days in association with 3 mg of norgestomet and 5 mg of VE (group IR/NG+VE; n=58) or reused Crestar®inserted during 8 days in association with 50mg of P4 and 2mg of BE (group IR/P4+BE; n=61). On the day the releasing devices were removed the animals received 7,5mg of Luprostiol and after 24 hours 1mg of BE. A fixed-time artificial insemination was done 54 to 56 hours after the removal of the releasing devices. Cows detected in estrus after the insemination at fixed-time were observed during a period of 49 days and reinseminated. The pregnancy diagnosis was done by ultrassonography 35 days after the IATF and after the end of the breeding season. The pregnancy rates of IATF (TP IATF) and at the end of the breeding season (TP EM) were estimated. There was no interaction between the characteristics of the (new or reused) releasing devices and the treatment given at the day the releasing devices were inserted (NG+VE e P4+BE). The use of new or used CIDR®had no effect on TP IATF (48,3% vs 48,7%) and on TP EM (85,2% vs 86,5%). The treatments with NG+VE e P4+BE did not have effect on TP IATF (49,5% vs 47,5%) and TP EM (86,5 vs 85,2%). There was a effect on the number of parturition (primipara and multipara) on the TP IATF (35% vs 52,7%; P<0,01) and on TP EM (71,9% vs 90,2%; P<0,01). It can be concluded that the new or once used releasing devices with norgestomet when associated with NG+VE or P4+BE promote highly satisfatory pregnancy rates in Nelore cows, being the better rates obtained in multipara females

Avaliação de bioensaios para a determinação da atividade biológica da gonadotrofina coriônica humana (hCG)

Biological activity of a given hormone is measured by its capacity to exert a specific, quantifiable biological effect. Aim of biological assays that measure activity of hCG is to construct prediction equations that associate increasing doses of hCG with changes in weights of genitalia, seminal vesicles and prostate gland in pre-pubertal male rats and weights of uterus and ovaries in pre-pubertal female mice. Objective of the present study was to evaluate efficiency of bioassays which used pre-pubertal male rats and female rats and mice to measure hCG activity. In experiment 1, male rats received 0, 4, 8 or 16IU of hCG daily, for 4 days. Prostate and seminal vesicles were weighed 24 hours after last injection. In experiment 2, female mice received 0, 3.33, 10, 33.33 or 100IU of hCG in one day. Ovaries and uteri were weighed 24 hours after the last injection. The hCG increased weights of genitalia in female mice. However, there were no satisfactory linear or quadratic associations between doses of hCG used and variables measured. It was concluded that assays tested showed only limited efficiency and sensibility to quantify hCG biological activity.A atividade biológica de um hormônio é mensurada pela sua capacidade em exercer um determinado efeito biológico que possa ser quantificado. Os ensaios biológicos in vivo que avaliam a atividade do hCG baseiam-se na sua capacidade de promover um aumento de peso do sistema genital, vesículas seminais e próstata em ratos impúberes e útero e ovários em camundongas impúberes. A partir destes efeitos, determinam-se equações relacionando as doses de hCG administradas com o aumento de peso do sistema genital. O objetivo do presente estudo foi avaliar a eficiência de bioensaios em mensurarem a atividade biológica do hCG utilizando ratos, ratas e camundongas impúberes. No experimento 1, ratos receberam 0, 4, 8 ou 16UI de hCG, a cada 24 horas, durante 4 dias. A próstata e/ou as vesículas seminais foram pesadas 24 horas após a última injeção. No experimento 2, camundongas receberam 0, 3,33, 10, 33,3 ou 100UI de hCG, em um único dia. Os ovários e/ou útero foram pesados 24 horas após a última injeção. O hCG promoveu aumento de peso do sistema genital de camundongas, entretanto, não houve associação linear ou quadrática significativa entre as doses de hCG utilizadas e os pesos das variáveis medidas, impossibilitando a determinação de equações confiáveis. Os protocolos testados, com as doses de hCG utilizadas demonstraram eficiência e sensibilidade limitadas para a quantificação da atividade biológica do hCG