Deciphering the functional role of EGR1 in Prostaglandin F2 alpha induced luteal regression applying CRISPR in corpus luteum of buffalo

BACKGROUND: PGF2α is essential for the induction of the corpus luteum regression which in turn reduces progesterone production. Early growth response (EGR) proteins are Cys2-His2-type zinc-finger transcription factor that are strongly linked to cellular proliferation, survival and apoptosis. Rapid elevation of EGR1 was observed after luteolytic dose of PGF2α. EGR1 is involved in the transactivation of many genes, including TGFß1, which plays an important role during luteal regression. METHODS: The current study was conducted in buffalo luteal cells with the aim to better understand the role of EGR1 in transactivation of TGFß1 during PGF2α induced luteal regression. Luteal cells from mid stage corpus luteum of buffalo were cultured and treated with different doses of PGF2α for different time durations. Relative expression of mRNAs encoding for enzymes within the progesterone biosynthetic pathway (3ßHSD, CYP11A1 and StAR); Caspase 3; AKT were analyzed to confirm the occurrence of luteolytic event. To determine if EGR1 is involved in the PGF2α induced luteal regression via induction of TGFß1 expression, we knocked out the EGR1 gene by using CRISPR/Cas9. RESULT: The present experiment determined whether EGR1 protein expression in luteal cells was responsive to PGF2α treatment. Quantification of EGR1 and TGFß1 mRNA showed significant up regulation in luteal cells of buffalo at 12 h post PGF2α induction. In order to validate the role of PGF2α on stimulating the expression of TGFß1 by an EGR1 dependent mechanism we knocked out EGR1. The EGR1 ablated luteal cells were stimulated with PGF2α and it was observed that EGR1 KO did not modulate the PGF2α induced expression of TGFß1. In PGF2α treated EGR1 KO luteal cell, the mRNA expression of Caspase 3 was significantly increased compared to PGF2α treated wild type luteal cells maintained for 12 h. We also studied the influence of EGR1 on steroidogenesis. The EGR1 KO luteal cells with PGF2α treatment showed no substantial difference either in the progesterone concentration or in StAR mRNA expression with PGF2α-treated wild type luteal cells. CONCLUSION: These results suggest that EGR1 signaling is not the only factor which plays a role in the regulation of PGF2α induced TGFß1 signaling for luteolysis.

Eficácia da associação dupla dose PGF2 alfa-eCG no proestro de vacas leiteiras mestiças submetidas à IATF / Efficacy of the double PGF2 alpha dose-eCG association in proestrus of crossbred dairy cows submitted to IATF

Objetivou-se avaliar o efeito de uma ou duas doses de prostaglandina F2α (PGF2α) associada ou não a gonadotrofina coriônica equina (eCG) sobre a dinâmica folicular, a função luteal pré-ovulatória, assim como as características morfofuncionais pós-ovulatórias do corpo lúteo (CL) em fêmeas mestiças cíclicas submetidas a um protocolo de inseminação artificial em tempo fixo (IATF). Para tanto, 29 vacas 3/4 Gir x Holandês multíparas foram submetidas ao exame de ultrassonografia (US) transretal e após a detecção do CL iniciou-se um protocolo de IATF em um dia denominado zero (D0), por meio da inserção do implante de progesterona (P4) associado à aplicação de 2,0mg de benzoato de estradiol. No D7 esses animais receberam 12,5mg de dinoprost trometamina. No D9 realizou a remoção dos dispositivos de P4 e aplicou 0,6mg de cipionato de estradiol. Nesse momento, as fêmeas foram subdivididas nos seguintes tratamentos: Grupo Controle (n=7), foi administrado 2,5mL de solução fisiológica; Grupo 2PGF (n=7), aplicou 12,5mg de dinoprost trometamina; Grupo eCG (n=7), administrou-se 300UI de eCG; Grupo 2PGF+eCG (n=8), realizou a aplicação de 300UI de eCG e 12,5mg de dinoprost trometamina. Para avaliar a dinâmica folicular foram realizados exames de US em modo B e power doppler (Mindray Z5, Shenzhen, China) a cada 12h do D7 até o momento da ovulação ou 96h após a remoção dos implantes de P4, mensurando-se o diâmetro folicular (DFOL), a área da parede folicular (AFOL) e a área de perfusão sanguínea da parede folicular (VFOL). Concomitante a cada exame, foram coletadas amostras de sangue sendo determinada a concentração sérica de P4 pré-ovulatória por meio da metodologia de quimioluminescência. No D24 foi realizada a US modo B e doppler analisando-se o diâmetro luteal (DCL), área luteal (ACL) e área de perfusão sanguínea do CL (VCL), assim como, foi coletada amostra de sangue para averiguar a concentração sérica de P4 pós-ovulatória. Os dados foram avaliados pelo Two-way ANOVA e análise de medidas repetidas considerando os efeitos do eCG, 2PGF e interação eCG*2PGF, P<0,05. Não houve diferença significativa entre os protocolos de sincronização para as variáveis DFOL, AFOL e VFOL ao longo do tempo da dinâmica folicular. Os grupos experimentais apresentaram uma concentração sérica de P4 pré-ovulatória semelhante em cada momento da avaliação. Não foi observada distinção da ACL e VCL entre os tratamentos hormonais, contudo o Grupo eCG demonstrou tendência (P=0,08) a apresentar maior DCL em relação ao Grupo 2PGF e 2PGF+eCG. Adicionalmente a estes achados, também foi constatado tendência (P=0,07) a maiores concentrações de progesterona no dia 24 do protocolo nos animais do Grupo eCG (11,00±3,32ng/mL) em relação ao Grupo 2PGF (6,37±1,31ng/mL), enquanto o Controle e 2PGF+eCG demonstraram resultados intermediários que se assemelham a ambos os grupos, com concentrações de 8,43±3,85 e 9,18±2,82ng/mL, respectivamente. As tentativas de ajustes no proestro foram incapazes de melhorar a qualidade folicular e minimizar a função luteal pré-ovulatória, assim como não incrementaram a morfologia do CL e a função luteal pós-ovulatória, sugerindo que em animais cíclicos mestiços protocolos de IATF com a utilização de uma única dose PGF2α e sem o suporte gonadotrófico da eCG parece promover adequada resposta folicular e luteal.(AU)

The study aimed to evaluate the effect of one or two prostaglandin doses F2α (PGF2a) with or without equine chorionic gonadotropin (eCG) in the follicular dynamics, the preovulatory luteal function, as well as the structural and functional characteristics post-ovulatory of the corpus luteum (CL) in cyclic crossbred females subjected to a fixed time artificial insemination (FTAI) protocol. For this, 29 multiparous 3/4 Gyr x Holstein cows were subjected to transrectal ultrasound examination (US) and upon detection of CL initiated a FTAI protocol on day called zero (D0) by the insertion of progesterone implant (P4) associated with the application of 2.0mg estradiol benzoate. On D7, these animals received 12.5mg of dinoprost tromethamine. At D9 happened the removal of the P4 devices and was applied 0.6mg of estradiol cypionate. At that time, the females were divided into the following treatments: control group (n=7) - which received 2.5mL of saline solution, 2PGF group (n=7) - received 12.5mg of dinoprost tromethamine, eCG group (n=7) - was administered 300IU eCG and eCG+2PGF group (n=8) - which received 300 IU eCG and 12.5mg of dinoprost tromethamine. To assess follicular dynamics were performed US scans B-mode and power doppler (Mindray Z5, Shenzhen, China) each 12h on D7 until the time of ovulation or until 96h after removal of the P4 implants, considering the follicular diameter (DFOL), the area of the follicular wall (AFOL) and the blood perfusion area of the follicular wall (VFOL). Concomitant with each test, blood samples were collected to determine the serum concentration of P4 preovulatory by chemiluminescence methodology. In D24 had held US B-mode and doppler to analyse the luteal diameter (DCL), luteal area (ACL) and blood perfusion area CL (VCL). Also, a blood sample was collected to determine the serum concentration of P4 post-ovulatory. All data was evaluated by Two-way ANOVA and repeated measures analysis considering the effects of eCG, 2PGF and eCG*2PGF, P<0.05. There was not significant difference between the synchronization protocols for DFOL, AFOL and VFOL variables over time of follicular dynamics. Experimental groups had a serum concentration of P4 preovulatory similar in every moment of evaluation. There wasn't distinction of ACL and VCL between hormone treatments. However, the eCG group showed a tendency (P=0.08) to present higher DCL compared to the 2PGF and 2PGF+eCG groups. In addition to these findings, there was also a tendency (P=0.07) to higher concentrations of P4 on D24 of the protocol in the animals of the eCG group (11.00±3.32ng/mL) compared to the 2PGF group (6,37±1.31ng/mL), meanwhile the Control and 2PGF+eCG showed intermediate results that resembled both groups, with concentrations of 8.43±3.85 and 9.18±2.82ng/mL, respectively. Attempts to adjust proestrus were unable to improve follicular quality and minimize preovulatory luteal function, nor did they increase CL morphology and post-ovulatory luteal function, suggesting that in cyclic animals, FTAI protocols using a single PGF2α dose and without the gonadotrophic support of eCG seems to promote adequate follicular and luteal responses.(AU)

Endometrial prostaglandin F2α in vitro production and its modulation regarding dominant follicle position in cattle / Produção in vitro de PGF F2α endometrial e sua modulação referente ao folículo dominante em bovinos

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.(AU)

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.(AU)

Role of interferon-tau in the maternal recognition of pregnancy / Atuação do Interferon-tau no reconhecimento materno da gestação / Papel del interferón tau en el reconocimiento materno de la gestación

Maternal recognition of pregnancy is a process by which the conceptus signals its presence to the mother in order to prolong the life of the corpus luteum (CL) thus maintaining the pregnancy. This process occurs between days 15 and 19 after fertilization and is the most important biological challenge for obtaining satisfactory reproductive indices in bovine. Interferon-tau (IFN-τ) glycoprotein -secreted in the uterus by the conceptus- has a paracrine action inhibiting the expression of estrogen receptors (ESR1) and oxytocin (OXTR) in the endometrium, thus preventing the release of luteolytic pulses of prostaglandin F2 alpha (PGF2α), hormone responsible for the onset of luteolysis. IFN-τ also increases the expression of several interferon-stimulated genes (ISGs) in the uterus, CL, and blood cells. Direct endocrine action of IFN-τ on extrauterine tissues stimulates ISGs expression, which in the corpus luteum seems to be involved with luteal resistance to luteolytic action of PGF2α. This review discusses recent findings on the luteolysis mechanism in the bovine and endocrine and paracrine mechanisms such as IFN-τ during the maternal recognition of pregnancy.

O reconhecimento materno da gestação é o processo pelo qual o concepto sinaliza sua presença à unidade materna, prolongando a vida do corpo lúteo (CL), determinando a manutenção da gestação. Esse processo que ocorre entre os dias 15 e 19 pós-fertilização, representa o desafio biológico mais importante para a obtenção de índices reprodutivos satisfatórios em bovinos. Nesta espécie, uma glicoproteína denominada Interferon-tau (IFN-τ), secretada pelo concepto no ambiente uterino, age de forma parácrina inibindo a expressão dos receptores de estrógenos (ESR1) e de ocitocina (OXTR) no endométrio, evitando a liberação de pulsos luteolíticos de prostaglandina F2 alfa (PGF2α), hormônio responsável pelo início da luteólise. O IFN-τ também aumenta a expressão de vários genes estimulados por interferons (ISGs) no útero, CL e em células sanguíneas. A ação endócrina direta do IFN-τ em tecidos extrauterinos estimula a expressão de ISGs, que no CL, parecem estar envolvidos com a resistência luteal à ação luteolítica da PGF2α. Portanto, esta revisão tem como objetivo discutir os achados recentes sobre o mecanismo de luteólise na espécie bovina, e a atuação parácrina e principalmente endócrina do IFN-τ, durante o período de reconhecimento materno da gestação.

El reconocimiento materno de la gestación es un proceso mediante el cual el concepto señaliza su presencia a la madre, prolongando la vida del cuerpo lúteo (CL), determinando el mantenimiento de la gestación. Este proceso que ocurre entre los días 15 y 19 después de la fertilización, representa el desafio biológico más importante para la obtención de índices reproductivos satisfactorios en bovinos. En esta especie, una glicoproteína denominada Interferon-tau (IFN-τ), es secretada por el concepto en el ambiente uterino, actúa de forma parácrina inhibiendo la expresión de los receptores de estrógenos (ESR1) y de oxitocina (OXTR) en el endometrio evitando la liberación de pulsos luteoliticos de prostaglandina F2 alfa (PGF2α), hormona responsable por el inicio de la luteólisis. El IFN-τ también aumenta la expresión de varios genes estimulados por interferon (ISGs) en el útero, CL y en las células sanguíneas. La acción endocrina directa del IFN-τ en tejidos extrauterinos estimula la expresión de ISGs, que en el cuerpo lúteo parece estar involucrados con la resistencia luteal a la acción luteolitica de la PGF2α. Por lo tanto esta revisión tiene como objetivo discutir los hallazgos recientes sobre el mecanismo de luteólisis en la especie bovina, así como el mecanismo paracrino y principalmente endocrino del IFN-τ durante el período de reconocimiento materno de la gestación.

Efecto del ácido linoléico sobre la producción de las prostaglandinas PGF2α y PGE2 en células endometriales / Linoleic acid effect on PGF2a and PGE2 prostaglandin production in endometrial cells

Objetivo. Evaluar el efecto in vitro del ácido linoléico sobre la producción de PGF2α y PGE2 en células endometriales epiteliales bovinas (CEEP). Materiales y métodos. Se cultivaron CEEP aisladas de tejido uterino y se suplementaron con AL a diferentes concentraciones (1 μM, 10 μM, 100 μM), oxitocina (OT) (0.1 μM) e interferón trofoectodérmico bovino (bINT-τ) (50 ng/ml). Se cuantificó la PGF2α y PGE2 a distintos tiempos (12, 24 y 36h). En el control, la PGF2α en el sobrenadante aumentó en el tiempo de cultivo y fue 1.2 veces mayor que la producción de PGE2. Resultados. El ácido linoléico disminuyó la concentración de PGF2α (p<0.05) en el sobrenadante, y no afectó (p>0.05) la producción de PGE2. El efecto conjunto de AL y OT sobre la producción de PGF2α difirió para cada uno de los tiempos; el ácido linoléico inhibió parcialmente el efecto estimulante de la OT sobre la producción de PGE2, el efecto conjunto del AL y el bINT-τ aumentó (p<0.05) esta inhibición hasta la hora 24. Conclusiones. El ácido linoléico afecta negativamente la concentración de PGF2α en el sobrenadante a través del tiempo. Respecto a la PGE2 se concluye que el ácido linoléico por sí solo no afecta la concentración en el sobrenadante.

Objective: Evaluate the in vitro effect of AL on the production of PGF2a and PGE2 in bovine epithelial endometrial cells (CEEP). Materials and methods. Isolated CEEP were cultured and supplemented with AL at different concentrations (1μM, 10μM, 100μM), oxytocin (OT) (0.1 μM) and bovine interferon (BINT-τ) (50 ng / ml). PGF2a and PGE2 were quantified as response variables at different times. In the control, the concentration of PGF2a in the supernatant increased over time and was 1.2 times greater than the production of PGE2. Results. AL reduced the concentration of PGF2a (p <0.05) in the supernatant, and did not affect (p> 0.05) the production of PGE2. The combined effect of LA and OT in the production of PGF2a is different in time, LA partially inhibited the stimulatory effect of OT in the production of PGE2, the joint effect of LA and BINT-τ increased (p<0.05) when this inhibition at 24h. Conclusions. LA alone negatively affects the concentration in the supernatant of PGE2 in time. Regarding PGE2 , LA alone does not affect the concentration in the supernatant.

Efecto del númeoo de aplicaciones de cloprostenl sobre la regresión de cuerposs lúteos, el retorno al celo y la ovulación, después de un tratamiento superovulatorio en donantes de embriones bovinos / The effectof nummber of clprostenol aplications on corpus luteum regressiion, return to estrus and ovulation, after a superovulatory treatment in bovine embryoddonors

El objetivo del presente trabajo fue evaluar la respuesta de la aplicación doble o triple de prostaglandina sobre la regresión de CL, ovulación y retorno al celo después de un tratamiento superovulatorio en vacas donantes de embriones. Para este experimento se tomaron 44 vacas asignadas al azar a dos grupos de tratamiento. Al primero (n = 23; CLO 0 & 7) se le aplicó una dosis de 500 μg de cloprostenol los días 0 (denominado día de la colecta) y 7; al segundo grupo (n = 21; CLO 0, 1 & 7) se le aplicó una inyección adicional el día 1. Los animales fueron examinados por ultrasonografía los días 0, 4, 7, 10 y 13 para evaluar la regresión de cuerpos lúteos y la ovulación. Se realizó observación del celo dos veces al día (mañana y tarde). El 60,8 y 52,3% de los grupos CLO 0 & 7 y CLO 0, 1 & 7, respectivamente, presentaron signos de celo. No se presentaron diferencias en el día del celo entre los grupos (7,2 ± 0,3, 7,3 ± 0,5 días). Tampoco hubo diferencia para la variable ovulación (8,5 ± 0,9 y 9,8± 0,9 días). Todas las vacas tratadas regresaron los cuerpos lúteos antes de finalizar el experimento. Cuando se analizó el tiempo de ovulación, se encontró que las vacas con 4 o menos cuerpos lúteos ovulan en un tiempo menor (P < 0,01) que las vacas con 5 o más cuerpos lúteos. Se concluye que no hay diferencia entre la doble o triple aplicación de cloprostenol para las variables. Sería innecesaria una aplicación adicional de cloprostenol el día 7.

The purpose of the present study was to evaluate the effect of prostaglandin (PG) treatment schedule on luteal regression and return to estrus in superovulated cows. A group of 44 donor cows were randomly assigned to two groups on the day of ova/ embryo collection (day 0). The first group (n = 23; CLO 0 & 7) was treated with 500 μg of cloprostenol on days 0 and 7 after embryo recovery; the second group (n = 21; CLO 0, 1 & 7) received an additional treatment with cloprostenol on day 1. All animals were scanned by transrectal ultrasonography on days 0 (embryo recovery), 4, 7, 10 and 13 to evaluate CL regression and subsequent ovulation. Estrus detection was done twice daily (am/pm) starting on day 4. There were no significant differences between groups in the percentage of animals returning to estrus (60.8% and 52.3%, respectively), or the intervals from the first PG treatment to estrus (7.2 ± 0.3 vs 7.3 ± 0.5 days), or ovulation (8.5 ± 0.9 vs 9.8± 0.9 days). All cows had luteolysis before the end of the experiment. Animals that had 4 CL or less ovulated earlier than cows that had 5 or more CL (P<0.01). In summary, there was no difference in luteal regression and the return to estrus and ovulation in donor cows treated with cloprostenol twice or three times after superovulation. It would not appear to be necessary to treat donors with PG 7 days later, since all cows had luteal regression after the first PG treatment.

Estradiol-17β altera expressão proteica endometrial em fêmeas bovinas tratadas no 17º dia do ciclo estral / 17β-estradiol alters endometrial protein expression in bovine females treated at the 17th day of estrous cycle

Em fêmeas bovinas, a liberação de prostaglandina F2α (PGF2α) é induzida in vivo pelo estradiol (E2). Acredita-se que o E2 estimule a síntese de proteínas essenciais na produção de PGF2α. Objetivou-se avaliar o efeito do E2 no incremento da concentração de proteínas totais e na modificação da composição proteica em explantes endometriais de fêmeas bovinas tratadas com E2 no 17º dia do ciclo estral. Novilhas cruzadas foram tratadas no 17º dia do ciclo estral, via intravenosa, com 0 mg (Grupo Controle; n = 6) ou 3 mg de E2 (Grupo E2; n = 6) e abatidas duas horas após. Explantes endometriais foram isolados, submetidos à extração de proteínas totais, quantificados e avaliados por Eletroforese Unidimensional em gel de poliacrilamida 10% SDS-PAGE. A concentração de proteínas totais não diferiu entre os grupos, 6296,10 + 439,90 µg/mL para o Grupo Controle e 8426,56 + 1156,00 µg/mL para o Grupo E2 (p = 0,1158). Não houve diferença significativa (p > 0,05) no perfil proteico dos explantes endometriais em géis corados com Coomasie Blue. Em géis corados com Nitrato de Prata verificou-se no Grupo E2 maior porcentagem relativa das bandas referentes ao peso molecular de 75 a 76 kDa (8,40% vs. 4,89%; no Grupo E2 e Controle respectivamente; p < 0,05) e 108 a 110 kDa (6,85% vs. 3,84%; no Grupo E2 e Controle respectivamente; p < 0,05). Observou-se no Grupo E2 menor porcentagem relativa da banda referente ao peso molecular de 90 kDa (5,78% vs. 9,83%; no Grupo E2 e Controle respectivamente; p < 0,05). Conclui-se que o E2 não incrementa a concentração de proteínas no endométrio, entretanto, altera a composição proteica nos explantes endometriais, indicando que o E2 altera a expressão de proteínas específicas.

In bovine females the release of prostaglandin F2α (PGF2α) is induced in vivo by estradiol (E2). It is believed that E2 stimulates the synthesis of essential proteins for the production of PGF2α. This study aimed to evaluate the effect of E2 in increasing the concentration of total protein and modifying the protein composition of endometrial explants from bovine females treated with E2 at the 17th day of estrous cycle. Crossbred heifers were treated at 17th day of estrous cycle intravenously with 0 mg (Control Group; n = 6) or 3 mg of E2 (E2 Group; n = 6) and killed two hours after. Endometrial explants were isolated, subjected to extraction of total protein, quantified and were analyzed by one-dimensional electrophoresis on polyacrylamide gel 10% SDS-PAGE. The concentration of total protein did not differ between groups, 6296.10 + 439.90 µg/mL for the Control Group and 8426.56 + 1156.00 µg/mL for E2 Group (p = 0.1158). There was no significant difference (p > 0.05) in the protein profile of endometrial explants in gels stained with Coomasie Blue. In gels stained with Silver Nitrate it was verified in E2 Group greater relative percentage of the bands referring to the molecular weight of 75 to 76 kDa (8.40% vs. 4.89% in E2 Group and Control respectively; p < 0.05) and 108 to 110 Kda (6.85% vs. 3.84% in E2 Group and Control respectively, p < 0.05). It was observed in E2 Group lower relative percentage of the band referring to the molecular weight of 90 kDa (5.78% vs. 9.83% in E2 Group and control respectively; p < 0.05). We concluded that the E2 does not increase the protein concentration in the endometrium, however, it modifies the proteinic composition in the endometrial explants, indicating that E2 alters the expression of specific proteins.

Effect of medroxy-progesterone acetate on follicular growth and endometrial cycloxygenase-2 (COX-2) expression during the bovine estrous cycle / Efeito do acetato de medroxi-progesterona sobre o crescimento filicular e expressão endometrial de ciclooxigenase-2 (COX-2) durante o ciclo estral de bovinos

The objective of this study was to evaluate the effect of medroxy-progesterone acetate (MAP) with or without estradiol benzoate (EB) on follicular growth during the estrous cycle in cattle. In the first experiment, Hereford cows were synchronized with a synthetic analogue of PGF2 alpha and were treated with two different doses of MAP (250 or 500 mg) with or without EB for 7 days starting on day 8 of the estrous cycle. Follicular growth was inhibited (P<0.05) in all cows except controls and those receiving 250mg MAP without EB. Seventy-five percent of the animals (15/20) showed estrus on days 21 and 22 of the cycle rather than at MAP withdrawal, demonstrating that these treatments did not induce estrus. To determine whether the EB treatment altered endometrial sensitivity to oxytocin and thus the luteolytic cascade, multiparous pre-synchronized cows received 5 mg of EB followed 6 hours later with 50 IU of oxytocin (OT; n=9). Eight hours after EB injection, endometrial fragments were collected from the cows on days 4, 13 and 17 of the estrous cycle and COX-2 gene expression was measured by PCR. EB increased COX-2 mRNA levels only on day 17 of the estrous cycle (P<0.05). In conclusion, MAP alone or associated with EB is able to suppress bovine follicular growth. However, EB in the presence of MAP is not efficient to induce luteolysis in cows when injected on day 8 of the estrous cycle.

Este estudo teve como objetivo avaliar o efeito do acetato de medroxi-progesterona (MAP) com ou sem benzoato de estradiol (BE) sobre o crescimento folicular durante o ciclo estral bovino. No primeiro experimento, vacas da raça Hereford foram sincronizadas com um análogo sintético de PGF2á e tratadas com duas doses diferentes de MAP (250 ou 500mg), com ou sem EB, durante 7 dias, iniciando-se no oitavo dia do ciclo estral. Observou-se uma inibição do crescimento folicular (P<0,05) em todas as vacas, exceto no grupo controle e no grupo que recebeu 250mg de MAP sem BE. Os 75 por cento dos animais não exibiu estro no momento da remoção do MAP, mas sim nos dias 21 e 22 do ciclo, demonstrando que os tratamentos não induziram cio. Para se determinar se o tratamento com BE alterou a sensibilidade endometrial à ocitocina e, assim, a cascata luteolítica, vacas multíparas pré-sincronizadas receberam 5mg de BE, seguidos, após 6 horas, de 50 UI de ocitocina (OT; n=9). Oito horas após a administração de BE, colheram-se fragmentos endometriais das vacas, nos dias 4, 13 e 17 do ciclo estral, mensurando-se a expressão gênica de COX-2 através de PCR. O BE aumentou os níveis de RNAm de COX-2 apenas no dia 17 do ciclo estral (P<0,05). Em conclusão, o MAP isolado ou associado a BE é capaz de suprimir o crescimento folicular bovino. Entretanto, o BE, na presença de MAP é ineficaz na indução da luteólise bovina, quando injetado no oitavo dia do ciclo estral.

Regresión prematura del cuerpo lúteo en ovejas en anestro tratadas con la hormona bovina del crecimiento / Premature luteal regression in anoestrus ewes treated with bovine somatotropin

In the first luteal phase of the transition period from anoestrus to cyclicity in the ewe, the secretion of prostaglandin F2 α (PGF2α) is bring forward and there is premature luteolysis. This early release of PGF2α is associated with the follicle properties that originate the corpus luteum.Therefore, if the preovulatory follicle produces more estrogens, the premature release of the PGF2α does not occur and the luteal phase is normal. On the other hand, bovine growth hormone (bST) induces follicular development and estradiol production. In this study it was assessed whether the administration of bST seven days before the induction of ovulation with hCG prevents premature luteal regression in anoestrus ewes. Suffolk ewes (n = 57) in seasonal anoestrus, determined through plasmatic progesterone concentrations, were used. Seven days before ovulation induction with 1 000 IU of hCG im, the ewes were randomly assigned to two groups: The bST group (n = 28) received 125mg of bST sc. The control group (n = 29) received physiological saline solution. Blood samples were taken daily from the day of hCG injection (day zero) until day 17 and the progesterone concentrations were determined by radioimmunoassay. Ninety six percent of the ewes from both groups (27/28, bST group and 28/29, control group) ovulated and formed a corpus luteum. The proportion of ewes with premature luteal regression was similar between treatments (bST = 37% vs control = 57%) (P = 0.135). The progesterone concentrations and the length of the luteal phase among the ewes that exhibited a corpus luteum of normal life span were also similar between groups (bST = 11.6 vs control = 11.1 days) (P > 0.05). It is concluded that the treatment with bST before ovulation induction with hCG in anoestrus ewes does not prevent the premature luteal regression.

En la primera fase lútea de la transición del anestro a la ciclicidad en la oveja, se adelanta la secreción de la prostaglandina F2 α (PGF2α) y ocurre la regresión prematura del cuerpo lúteo. La liberación anticipada de la PGF2α se asocia con las características del folículo que da origen al cuerpo lúteo; así, cuando el folículo produce más estrógenos, la liberación de la PGF2α no se adelanta y la fase lútea es normal. En este contexto, la hormona bovina del crecimiento (bST) estimula el desarrollo folicular y la producción de estradiol. En este estudio se probó si la administración de la bST, siete días antes de la inducción de la ovulación con hCG en ovejas en anestro, evita la regresión prematura del cuerpo lúteo. Se utilizaron 57 ovejas de la raza Suffolk en anestro estacional, lo cual se comprobó mediante la medición de las concentraciones plasmáticas de progesterona. Siete días antes de la inducción de la ovulación con 1 000 UI de hCG im, las ovejas se asignaron al azar en dos grupos: el grupo bST (n = 28) recibió 125 mg de bST sc; el grupo testigo (n = 29) recibió solución salina fisiológica. Se obtuvieron muestras de sangre diariamente a partir del día de la aplicación de hCG (día cero) hasta el día 17 y se determinaron las concentraciones de progesterona mediante radioinmunoanálisis. En ambos grupos se registró 96% de ovulación y formación de cuerpo lúteo (27/28, grupo bST y 28/29, grupo testigo). La proporción de ovejas que presentaron regresión prematura del cuerpo lúteo fue similar (P = 0.135) entre los tratamientos (bST = 37% vs testigo = 57%). Las concentraciones de progesterona y la duración de la fase lútea en las ovejas que tuvieron cuerpos lúteos de vida normal (bST = 11.6 días vs testigo = 11.1 días) fueron similares entre los grupos (P > 0.05). No se encontró evidencia que el tratamiento con bST antes de la inducción de la ovulación con hCG en ovejas en anestro evita la regresión prematura del cuerpo lúteo.

Efeito da frequência de amamentação sobre a taxa de ovulação após tratamento com desmame temporário + GnRH, sobre a incidência de luteólise prematura no primeiro ciclo estral pós-parto e sobre o desempenho da progênie de vacas Nelore / Effect of suckling frequency on ovulation rate after temporary weaning + GnRH treatment, incidence of premature luteolysis in the first postpartum estrous cycle, and performance of calves from Nelore cows

Avaliou-se o efeito da restrição na frequência de amamentação sobre o diâmetro folicular no dia 0 (DFOL), sobre a taxa de ovulação (TO), e sobre a incidência de luteólise prematura no primeiro ciclo estral pós-parto (ILP) de vacas Nelore multíparas, em anestro, submetidas à amamentação ad libitum (controle; n= 115) ou amamentação uma vez ao dia (restrito; n= 109), entre os dias -14 e 9 do experimento, e estudou-se o efeito desses tratamentos sobre o peso à desmama da progênie dessas vacas. Induziu-se ovulação com remoção de bezerros entre os dias -2 e 0 e aplicação de 100μg de GnRH no dia 0. Somente animais que ovularam foram mantidos no experimento (n= 125). A ocorrência de luteólise prematura foi avaliada por meio da dosagem da concentração sérica de progesterona nos dias 5 e 9. A TO não foi influenciada pelos tratamentos (55,8 por cento; P>0,1), e as vacas do tratamento restrito apresentaram maior DFOL (10,90±0,26 vs. 10,18±0,21mm; P<0,05) e menor ILP (21,4 por cento vs. 43,5 por cento; P<0,05). Os bezerros do tratamento controle foram mais pesados (162,32±2,08 vs. 155,91±4,12kg; P<0,05). Conclui-se que a restrição na frequência de amamentação em vacas Nelore reduz a ILP, porém com possível efeito negativo no desenvolvimento dos bezerros.

The effects of restricted suckling on follicular diameter at day 0 (FDDO), ovulation rate (OR), and incidence of premature luteolysis in the first post partum estrous cycle (PLI) of Nelore cows and the effects on weight at weaning (WW) of progenie of these cows were evaluated. Multiparous anestrous postpartum Nelore cows were submitted to ad libitum suckling (control; n= 115) or once-a-day suckling (restricted; n= 109) from days -14 to 9 of the experiment. For both treatments, a temporary calf removal was performed from days -2 to 0, and on day 0 cows received 100μg of GnRH. Only animals that ovulated after GnRH treatment were used in the experiment (n= 125). The occurrence of premature luteolysis was evaluated by dosage of serum progesterone concentrations on days 5th and 9th. The OR was not affected by treatments (55.8 percent; P>0.1), but cows from restricted treatment had larger FDDO at time of GnRH treatment (10.90±0.26 vs. 10.18±0.21mm; P<0.05) and lower PLI (21.4 percent vs. 43.5 percent; P<0.05) than cows from control treatment. Calves from control treatment had higher WW than calves from restricted treatment (162.32±2.08 vs. 155.91±4.12kg; P<0.05). These results indicate that the restriction in frequency of suckling in Nelore cows may decrease the incidence of premature luteolysis in the first postpartum estrous cycle; however, with possible negative effects on calves development.

Mecanismos da luteólise: revisão de literatura / Mechanisms of luteolysis: article review / Mecanismos de luteólisis: revisión de literatura

O principal papel do corpo lúteo é a produção de progesterona. Uma adequada função luteal é crucial para determinar a duração do ciclo estral ou a manutenção e sucesso de uma possível gestação. O crescimento do corpo lúteo só é comparado ao de tumores invasivos; e sua regressão se dá em poucos dias, por meio de um processo chamado luteólise, que é desencadeado pela secreção de prostaglandina F2alfa (PGF2α). O conhecimento dos fatores ligados ao processo de regulação do corpo lúteo é importante para a manipulação do ciclo estral, na tentativa de aumentar a eficiência reprodutiva dos animais domésticos. Conhecimentos em relação a esse processo podem mudar a metodologia de pesquisa e, principalmente, a aplicação prática de procedimentos, visando o controle do ciclo estral. Nesse contexto, este estudo tem como objetivo apresentar informações atuais sobre a luteólise, que envolvem hipóteses sobre o fluxo sanguíneo uterino e ovariano, além do processo de morte celular por apoptose.

The main role of the corpus luteum is the progesterone production and adequate luteal function is crucial to determine the duration of oestrous cycle or continuation and success of a possible pregnancy. The corpus luteum growth is compared to most invasive tumors and its regression occurs in a few days in the process called luteolysis, triggered by the secretion of prostaglandin F2alfa (PGF2 α). The knowledge of factors associated with the process of corpus luteum regulating is important for the reproductive cycle manipulation in an attempt to increase the reproductive efficiency of livestock animals. New awareness regarding the process can change radically the methodology of research and the implementation practice ofthe procedures for oestrous cycle control. The aim of this review is to bring information about the new theories of luteolysis, which involve assumptions on the uterine and luteal blood flow and the process of cell death by apoptosis.

El principal papel del cuerpo lúteo es la producción de la progesterona. Una adecuada función lútea es fundamental para determinar la duración del ciclo estral o la manutención y el éxito de una posible preñez. El crecimiento del cuerpo lúteo solo se compara al de tumores invasores; y su regresión se produce en pocos días, por medio de un proceso llamado luteólisis, provocado por la secreción de prostaglandina F2alfa (PGF2α). El conocimiento de los factores asociados al proceso de regulación del cuerpo lúteo es importante para la manipulación del ciclo estral, con la intención de aumentar la eficiencia reproductiva de los animales domésticos. Conocimientos en relación a ese proceso pueden cambiar la metodología de la investigación y, principalmente, buscar el control del ciclo estral. En ese contexto, la revisión tuvo como objetivo presentar informaciones actuales sobre la luteólisis, que implican hipótesis sobre el flujo sanguíneo uterino y ovárico, además del proceso de muerte celular por apoptosis.

Alternative low doses and routes of administering a prostaglandin F2alpha analogue to induce luteolysis in Nelore cows

The present study was conducted in order to verify the efficacy of lower doses and alternative routes of a prostaglandin F2 alpha analogue, luprostiol (PGF), for the induction of luteolysis and the precipitation of estrus in nonlactating Nelore cows (Bos taurus indicus). A conventional dose (15 mg) of PGF was compared to doses lower than the conventional dose, which ranges from 10 to 50%, that were administered intramuscularly (IM), intravulvosubmucosally (IVSM), or in the Bai-hui acupuncture site located within the lumbosacral area. The cows were administered PGF 8 day after estrus in the presence of a corpus luteum, and randomly assigned to the following groups: G1 (positive control), 15 mg, IM (n = 23); G2, 7.5 mg, IM (n = 23); G3, 3.75 mg, IM (n = 24); G4, 7.5 mg, IVSM (n = 25); G5, 3.75 mg, Bai-hui acupoint (n = 24); and G6, 1.5 mg, Bai-hui acupoint (n = 25). The results indicated that 50% of a conventional dose of PGF (7.5 mg) resulted in a complete luteal regression (plasma progesterone <1 ng/ml) at Hour 48, and hastened estrus, regardless of whether or not PGF was administered IM or IVSM. Comparatively, 10 or 25% of the conventional dose, even when administered to the Bai-hui acupoint, resulted in an initial reduction in the concentration of progesterone at Hour 24, followed by an increase observed at Hour 48. In conclusion, 25% of a conventional PGF dose administered via the Bai-hui acupoint proved inadequate to induce a complete luteal regression, whereas 50% of a conventional dose administered IM or IVSM was found to be the minimal dose required to induce effectively a complete luteal regression, and to precipitate the onset of estrus in nonlactating Nelore cows.

Regressão luteal e dinâmica folicular após luteólise natural ou induzida por cloprostenol em vacas da raça Gir

The luteal regression and the follicular dynamic were evaluated during the natural luteal regression period (n=14) or after induction of luteolysis by the administration of 500 g of cloprostenol (n=13), using a portable ultrasound device. Luteolysis induction increased luteal regression over 24 (0.89±0.13×0.24±0.17cm²/day; P 0.05) and 48 hours period (0.78±0.15×0.36±0.07cm²/day; P 0.05), but the reduction of progesterone concentration was similar (P>0.05). There was no difference (P>0.05) in follicular dynamic between the two groups. Cows in which the largest follicle during luteolysis was the ovulatory follicle presented shorter periods of follicular growth (3.71±0.56×5.26±0.34 days; P 0.05) and luteolysis to estrous intervals (85.71±14.68×121.33±8.34 hours; P 0.05). This study shows that functional (but not morphological) regression of corpus luteum and follicular dynamics after spontaneous or induced luteolysis are similar in Gir cattle.

A regressão luteal e a dinâmica folicular foram avaliadas durante o período de regressão luteal natural (n=14) ou após a indução artificial da luteólise pela aplicação de 500 g de cloprostenol (n=13), utilizando-se um aparelho portátil de ultra-som. Após a indução da luteólise foi detectada maior taxa de regressão luteal em 24 (0,89± 0,13×0,24± 0,17cm²/dia; P 0,05) e 48 horas (0,78±0,15×0,36±0,07cm²/dia P 0,05), porém a redução na concentração de progesterona foi semelhante (P>0,05). Não houve diferença (P>0,05) nas características da dinâmica folicular entre os dois grupos. No momento da luteólise, quando havia um folículo dominante funcional, observou-se redução na duração do crescimento folicular (3,71±0,56×5,26±0,34 dias; P 0,05) e no intervalo luteólise-estro (85,71±14,68×121,33±8,34 horas; P 0,05). Os resultados demonstram que a regressão funcional do corpo lúteo e a dinâmica folicular são semelhantes após a luteólise natural ou induzida em vacas da raça Gir, e que o fator determinante no intervalo luteólise-estro é o estádio fisiológico dos folículos presentes.

Extra-pituitary action of gonadotropin releasing hormone: possible application.

Chronic administration of a potent gonadotropin releasing hormone inhibits ovulation in women. The suppression of gonadal function during long term treatment with the GnRH analogues is ascribable to inhibition of gonadotropin secretion caused by the down regulatory action of the decapeptide at the pituitary level. Reduced progesterone production with premature onset of menstruation has been observed in women injected with the agonist during the midluteal phase. The decapeptide however, has no effect on in vitro human ovarian steroidogenesis. Specific receptors for GnRH have been located on rodent ovarian cells, but corpora lutea of rhesus monkey and human ovaries seem to lack these receptors. The luteolytic effect in women thus appears to be central in origin and not a direct effect on the corpus luteum. Recently, a superactive agonist of GnRH given around the peri-implantation period has been shown to terminate pregnancy in baboons. Monoclonal antibodies against GnRH administered during the same period in a fertile cycle also abrogated pregnancy in these animals. Using immuno-enzymatic techniques GnRH has been localized on the placenta. GnRH also exerts a stimulatory effect on hCG production by the placental villi maintained in culture. Addition of anti-luteinizing hormone releasing hormone antibodies blocks this effect completely. It seems that placenta is the only other tissue besides the pituitary where GnRH has probably a regulatory role in the human female.

In vitro responsiveness of hamster corpora lutea undergoing luteolysis to luteinizing hormone.

Corpora lutea removed from pregnant hamster deprived of endogenous luteinizing hormone for varying periods were compared for their responsiveness to externally added luteinizing hormone. The corpora lutea removed on the 8th day of pregnancy exhibited a dose-dependent increase in progesterone production in response to added luteinizing hormone upto a concentration of 2·5 μg/ml. The total progesterone synthesised by the corpora lutea decreased with increase in the duration of in vivo luteinizing hormone deprivation. However, the hormone deprivation had to be for a minimum period of 24 h before a marked reduction in the in vitro responsiveness could be seen. Neutralisation of endogenous luteinizing hormone increased the luteal cholesterol ester concentration, while in vitro incubation of such tissue with luteinizing hormone resulted in a marked reduction in cholesterol ester levels. Corpora lutea removed from hamsters on day 8, 15 and 16 of pregnancy when compared for their responsiveness in vitro to added luteinizing hormone showed that the luteal tissue of day 8 produced more progesterone relative to those of day 15/16. In contrast, depletion of free and esterified cholesterol increased with the increase in age of corpora lutea (from 15% on day 8 to 67% on day 16).