Neospora caninum DNA distribution in tissues of gerbils as experimental models of chronic neosporosis / [Distribuição do DNA de Neospora caninum em tecidos de gerbilos como modelos experimentais de neosporose crônica]

Neospora caninum is the main etiologic agent of neosporosis in domestic animals and its pathogenesis comprises two characteristic phases: acute and chronic. Rodents are used as experimental models to mimic acute and chronic bovine neosporosis. In this study, we inoculated a total of 27 female gerbils, with different doses of N. caninum tachyzoites aiming to induce chronic disease. DNA was extracted from different organs of each animal after spontaneous death or euthanasia. Encephalic tissues were submitted to a highly sensitive real time PCR aiming to detect chronically infected animals. All the other samples were submitted to standard PCR. A total of 11 gerbils died due to acute neosporosis, as confirmed by N. caninum DNA detection in organs. 5x103 tachyzoites/mL of N. caninum was the dosage of antigen that can induce chronic infection in gerbils. In the encephalon sections of some animals that showed clinical signs of persistent infection, we found 70% positive for the anterior encephalon section, suggesting this area as preferential for cyst formation. Therefore, we determined the doses of tachyzoites that cause acute or chronic infection and detection of positive tissues, preferably, systemic organs during acute and encephalon in chronic phases.(AU)

Neospora caninum é o principal agente etiológico da neosporose em animais domésticos, e sua patogênese compreende duas fases características: aguda e crônica. Roedores são usados como modelos experimentais para simular neosporose bovina aguda e crônica. Neste estudo, foi inoculado um total de 27 gerbilos, fêmeas, com diferentes doses de taquizoítos de N. caninum, visando induzir doença crônica. O DNA foi extraído de diferentes órgãos de cada animal após a morte espontânea ou a eutanásia. Os tecidos encefálicos foram submetidos à PCR em tempo real de alta sensibilidade para detecção de animais com infecção crônica. Todas as outras amostras foram submetidas à PCR padrão. Um total de 11 gerbilos morreu devido à neosporose aguda, como confirmado pela detecção de DNA de N. caninum nos órgãos. A dosagem de antígeno que pode induzir infecção crônica foi de 5x103 taquizoítos/mL de N. caninum. Em seções do encéfalo de alguns animais, que apresentaram sinais clínicos de infecção persistente, encontraram-se 70% de positividade para a seção do encéfalo anterior, sugerindo essa área como preferencial para a formação de cisto. Assim, foram determinadas,, em gerbilos, as dosagens de taquizoítos capazes de induzir infecção crônica ou aguda, bem como foram detectados tecidos positivos, preferencialmente, em órgãos sistêmicos, na fase aguda, e no encéfalo, na crônica.(AU)

The differentiation potential of adipose tissue-derived mesenchymal stem cells into cell lineage related to male germ cells / Potencial de diferenciação de células-tronco mesenquimais derivadas do tecido adiposo em células da linhagem germinativa masculina

The adipose tissue is a reliable source of Mesenchymal stem cells (MSCs) showing a higher plasticity and transdifferentiation potential into multilineage cells. In the present study, adipose tissue-derived mesenchymal stem cells (AT-MSCs) were isolated from mice omentum and epididymis fat depots. The AT-MSCs were initially compared based on stem cell surface markers and on the mesodermal trilineage differentiation potential. Additionally, AT-MSCs, from both sources, were cultured with differentiation media containing retinoic acid (RA) and/or testicular cell-conditioned medium (TCC). The AT-MSCs expressed mesenchymal surface markers and differentiated into adipogenic, chondrogenic and osteogenic lineages. Only omentum-derived AT-MSCs expressed one important gene marker related to male germ cell lineages, after the differentiation treatment with RA. These findings reaffirm the importance of adipose tissue as a source of multipotent stromal-stem cells, as well as, MSCs source regarding differentiation purpose.(AU)

O tecido adiposo é uma fonte apropriada de células-tronco mesenquimais (MSCs), as quais demonstram ampla plasticidade com capacidade de transdiferenciar em diversas linhagens. No presente estudo, as células-tronco mesenquimais derivadas do tecido adiposo (AT-MSC) foram isoladas de tecido adiposo localizado nas regiões próximas ao omento e testículos de camundongos. Primeiramente, as AT-MSCs foram comparadas com base na expressão de marcadores antigênicos de superfície e no potencial de diferenciação nas três linhagens mesodérmicas. Além disso, AT-MSC, de ambas as fontes, foram cultivadas com meio de diferenciação contendo ácido retinóico (RA) e / ou meio condicionado testicular (TCC). As AT-MSCs expressaram marcadores de superfície mesenquimais e diferenciaram nas linhagens adipogênica, condrogênica e osteogênica. Após o tratamento com RA, somente as AT-MSCs isoladas do tecido adiposo depositado na região do omento expressaram um único importante marcador relacionado às células da linhagem germinativa masculina. Estes resultados reafirmam a importância do tecido adiposo como fonte de células-tronco estromais-multipotentes, bem como, uma fonte de MSCs para estudos de diferenciação.(AU)

The differentiation potential of adipose tissue-derived mesenchymal stem cells into cell lineage related to male germ cells / Potencial de diferenciação de células-tronco mesenquimais derivadas do tecido adiposo em células da linhagem germinativa masculina

The adipose tissue is a reliable source of Mesenchymal stem cells (MSCs) showing a higher plasticity and transdifferentiation potential into multilineage cells. In the present study, adipose tissue-derived mesenchymal stem cells (AT-MSCs) were isolated from mice omentum and epididymis fat depots. The AT-MSCs were initially compared based on stem cell surface markers and on the mesodermal trilineage differentiation potential. Additionally, AT-MSCs, from both sources, were cultured with differentiation media containing retinoic acid (RA) and/or testicular cell-conditioned medium (TCC). The AT-MSCs expressed mesenchymal surface markers and differentiated into adipogenic, chondrogenic and osteogenic lineages. Only omentum-derived AT-MSCs expressed one important gene marker related to male germ cell lineages, after the differentiation treatment with RA. These findings reaffirm the importance of adipose tissue as a source of multipotent stromal-stem cells, as well as, MSCs source regarding differentiation purpose.(AU)

O tecido adiposo é uma fonte apropriada de células-tronco mesenquimais (MSCs), as quais demonstram ampla plasticidade com capacidade de transdiferenciar em diversas linhagens. No presente estudo, as células-tronco mesenquimais derivadas do tecido adiposo (AT-MSC) foram isoladas de tecido adiposo localizado nas regiões próximas ao omento e testículos de camundongos. Primeiramente, as AT-MSCs foram comparadas com base na expressão de marcadores antigênicos de superfície e no potencial de diferenciação nas três linhagens mesodérmicas. Além disso, AT-MSC, de ambas as fontes, foram cultivadas com meio de diferenciação contendo ácido retinóico (RA) e / ou meio condicionado testicular (TCC). As AT-MSCs expressaram marcadores de superfície mesenquimais e diferenciaram nas linhagens adipogênica, condrogênica e osteogênica. Após o tratamento com RA, somente as AT-MSCs isoladas do tecido adiposo depositado na região do omento expressaram um único importante marcador relacionado às células da linhagem germinativa masculina. Estes resultados reafirmam a importância do tecido adiposo como fonte de células-tronco estromais-multipotentes, bem como, uma fonte de MSCs para estudos de diferenciação.(AU)

Expression of growth and differentiation Factor 9 and cognate receptors during final follicular growth in cattle

Mutations in growth and differentiation factor9 (GDF9) gene are associated to sterility or,paradoxically, increased ovulation rate in ewes. Despiteits importance, the exact function of GDF9 in ovarianphysiology is still poorly understood. This study aimedto investigate GDF9 function during dominant folliclegrowth and its regulation in follicular fluid. Theregulation of GDF9 receptors in GnRH/LH-stimulatedgranulosa cells was also investigated. In a firstexperiment, a new follicular wave was induced and theintrafollicular GDF9 treatment into the largest growingfollicle (8.5-9.5 mm) at both 100 (n = 3) and 1000ng/ml(n = 4) had no effect on follicular growth, estrusmanifestation and ovulation compared to control (PBSinjected)follicles (n = 3). In a second experiment,follicles were obtained just after follicular deviation(day 4 after follicular emergence) and the abundance ofGDF9 in follicular fluid did not differ between healthydominant (n = 4) and atretic subordinate follicles (n =4), as assessed by western blot analysis. Finally, mRNAexpression of BMPR2 and TGFBR1 receptors wasevaluated in granulosa cells obtained from preovulatoryfollicles (>12 mm diameter) obtained 0, 3, 6, 12 or 24 hafter i.m. GnRH administration (n = 4-5follicles/moment). Both receptors were significantly upregulated 12 h after GnRH treatment. Present results donot confirm the hypothesis that GDF9 inhibits dominantfollicle growth and suggests a minor role in determiningfollicle fate. In the other hand, GDF9 receptorsregulation in GnRH/LH-stimulated granulosa cellsprovides the first in vivo evidence of its involvement inthe complex cascade of events that culminates inovulation and luteinization in cattle.(AU)

Expression of growth and differentiation Factor 9 and cognate receptors during final follicular growth in cattle

Mutations in growth and differentiation factor9 (GDF9) gene are associated to sterility or,paradoxically, increased ovulation rate in ewes. Despiteits importance, the exact function of GDF9 in ovarianphysiology is still poorly understood. This study aimedto investigate GDF9 function during dominant folliclegrowth and its regulation in follicular fluid. Theregulation of GDF9 receptors in GnRH/LH-stimulatedgranulosa cells was also investigated. In a firstexperiment, a new follicular wave was induced and theintrafollicular GDF9 treatment into the largest growingfollicle (8.5-9.5 mm) at both 100 (n = 3) and 1000ng/ml(n = 4) had no effect on follicular growth, estrusmanifestation and ovulation compared to control (PBSinjected)follicles (n = 3). In a second experiment,follicles were obtained just after follicular deviation(day 4 after follicular emergence) and the abundance ofGDF9 in follicular fluid did not differ between healthydominant (n = 4) and atretic subordinate follicles (n =4), as assessed by western blot analysis. Finally, mRNAexpression of BMPR2 and TGFBR1 receptors wasevaluated in granulosa cells obtained from preovulatoryfollicles (>12 mm diameter) obtained 0, 3, 6, 12 or 24 hafter i.m. GnRH administration (n = 4-5follicles/moment). Both receptors were significantly upregulated 12 h after GnRH treatment. Present results donot confirm the hypothesis that GDF9 inhibits dominantfollicle growth and suggests a minor role in determiningfollicle fate. In the other hand, GDF9 receptorsregulation in GnRH/LH-stimulated granulosa cellsprovides the first in vivo evidence of its involvement inthe complex cascade of events that culminates inovulation and luteinization in cattle.

Avaliações clínica, ecográfica e anatomofisiológica do alotransplante parcial de vesícula urinária com células-tronco mesenquimais alogênicas derivadas do tecido adiposo em coelhos / Clinical, sonographic, anatomic and physiological assessments of partial allotransplant urinary bladder with allogeneic mesenchymal stem cells derived from adipose tissue in rabbits

Os problemas relacionados ao armazenamento vesical são muitos e relevantes. Eles, além de influírem de forma efetiva na qualidade de vida, podem eventualmente evoluir para falência renal. Existem vários trabalhos, os quais descrevem as propriedades imunomoduladoras e imunossupressoras das células-tronco mesenquimais derivadas do tecido adiposo (ADSCs). Objetiva-se com o presente avaliar clínica, ecográfica e anatomofisiologicamente o alotransplante parcial de bexiga a fresco em coelhos, utilizando como agente imunomodulador ADSCs alogênicas. Para isso foram utilizados 25 coelhos, sendo um deles macho e doador das ADSCs, e os outros 24 eram fêmeas, submetidas a alotransplante parcial de bexiga, sendo tratadas com ciclosporina (GCi) ou células-tronco mesenquimais (GCe). Conclui-se que as ADSCs foram suficientes para evitar sinais clínicos e ecográficos de rejeição ao alotransplante de vesícula urinária, mantendo a estrutura anatomofisiológica vesical por até 30 dias em coelhos.(AU)

The problems related to bladder storage are many and significant. In addition to effectively impacting the quality of life, they can eventually progress to kidney failure. There are several studies which describe the immunomodulatory and immunosuppressive properties of ADSCs. The aim of this study is to clinically, through sonography, anatomically and physiologically evaluate fresh partial allograft bladder from rabbits using allogeneic ADSCs as immunomodulator agents. For such, 25 rabbits were used, one being a male ADSCs donor, and the other 24 females who underwent simultaneous partial allograft bladder, being treated with cyclosporine (GCi) or mesenchymal stem cells (GCe). It was concluded that ADSCs were sufficient to prevent clinical and ultrasound signs of allograft rejection of the urinary bladder. These bladders retained the anatomophysiological structure for 30 days in rabbits.(AU)

Participação da esfingosina 1-fosfato e do fator inibidor da leucemia no cultivo in vitro de folículos ovarianos pré-antrais de cabras / The role of Sphingosine 1-phosphate and Leukemia Inhibitory Factor in the culture of goats preantral follicles in vitro

A manipulação de oócitos inclusos em folículos ovarianos pré-antrais (MOIFOPA) possibilita oaumento do potencial reprodutivo das fêmeas a partir do isolamento e cultivo de folículos pré-antrais (FOPA).Fisiologicamente, vários fatores de crescimento estão envolvidos durante o processo da foliculogênese, muitosdos quais até o momento não foram testados em cabras. Esta revisão tem como objetivo correlacionar aparticipação da S1P e do LIF no cultivo dos FOPA e, assim, possibilitar uma melhor compreensão dosmecanismos relacionados com a foliculogênese.(AU)

The manipulation of oocytes included in preantral Follicles increasing the reproductive potential of thefemales from the isolation and culture of preantral follicles was studied. Physiologically various growth factorsare involved in the folliculogenesis process, many of which have not been tested in goats so far. This review aimsto correlate the involvement of S1P and LIF in the culture of preantral follicle enabling a better understandingof the mechanisms related to folliculogenesis.(AU)

Participação da esfingosina 1-fosfato e do fator inibidor da leucemia no cultivo in vitro de folículos ovarianos pré-antrais de cabras / The role of Sphingosine 1-phosphate and Leukemia Inhibitory Factor in the culture of goats preantral follicles in vitro

A manipulação de oócitos inclusos em folículos ovarianos pré-antrais (MOIFOPA) possibilita oaumento do potencial reprodutivo das fêmeas a partir do isolamento e cultivo de folículos pré-antrais (FOPA).Fisiologicamente, vários fatores de crescimento estão envolvidos durante o processo da foliculogênese, muitosdos quais até o momento não foram testados em cabras. Esta revisão tem como objetivo correlacionar aparticipação da S1P e do LIF no cultivo dos FOPA e, assim, possibilitar uma melhor compreensão dosmecanismos relacionados com a foliculogênese.

The manipulation of oocytes included in preantral Follicles increasing the reproductive potential of thefemales from the isolation and culture of preantral follicles was studied. Physiologically various growth factorsare involved in the folliculogenesis process, many of which have not been tested in goats so far. This review aimsto correlate the involvement of S1P and LIF in the culture of preantral follicle enabling a better understandingof the mechanisms related to folliculogenesis.

Sphingosine 1-phosphate promotes activation of aprine preantral follicle in vitro / Esfingosina 1-fosfato promove ativação de folículos pré-antrais de caprinos in vitro

This study describes the effect of sphingosine 1-phosphate (S1P) for development of preantral follicle, therefore the activation and follicular viability of caprine follicles cultured in vitro. Ovarian fragments were cultured for 1 or 7 days in Minimum Essential Medium with different S1P concentrations (0, 1, 10, 50, 100 or 200ng/mL). All ovarian fragments were processed for histological analysis in optical microscopy, transmission electron microscopy and fluorescence analysis. The treatment using 1ng/mL of S1P was able to maintain the percentage of normal follicles with the progression of the culture from day 1 to 7. At end of the 7-day culture period there was a significant reduction (P<0.05) in the percentage of primordial follicles in all groups treated with S1P, compared with fresh control (FC) and Control Culture (CC), which was followed by an increase of activated follicles (intermediary, primary and secondary). In addition, the culture for 7 days with media supplemented with S1P with 1ng/mL preserved the ultrastructure of organelles and kept the preantral follicular viability when evaluated by fluorescence microscopy. In conclusion, after 7 days of culture, the 1ng/mL of S1P activates the development of preantral caprine follicles, cultured in situ and maintains the oocitary and follicular viability.(AU)

Este estudo descreve o efeito da esfingosina 1-fosfato (S1P) no desenvolvimento de folículos pré-antrais, portanto da ativação e viabilidade de folículos caprinos cultivados in vitro. Fragmentos de ovários foram cultivados por um ou sete dias em meio essencial mínimo com diferentes concentrações de S1P (0, 1, 10, 50, 100 ou 200ng/mL). Os fragmentos de ovário foram processados para análise histológica em microscopia óptica, microscopia eletrônica e microscopia de fluorescência. O tratamento usando 1ng/mL de S1P foi capaz de manter a porcentagem de folículos normais durante o período de cultivo de sete dias. Ao final do período de cultivo, houve uma redução significativa (p<0,05) na porcentagem de folículos primordiais em todos os grupos tratados com S1P, comparados com os grupos controle (FC e CC), seguida por um aumento do número de folículos ativados (intermediários, primários e secundários). Adicionalmente, na cultura por sete dias com meio suplementado com S1P (1ng/mL), houve preservação da ultraestrutura das organelas e manteve-se a viabilidade dos folículos pré-antrais avaliados por microscopia de fluorescência. Em conclusão, após sete dias de cultura, o meio suplementado com 1ng/mL de S1P ativa o desenvolvimento de folículos pré-antrais de caprino, cultivados in situ e mantém as viabilidades oocitária e folicular.(AU)

Avaliação do emprego do eCG em um programa hormonal de indução/sincronização de estro e ovulação em novilhas de corte entre 12 e 14 meses de idade / Evaluation of the use of eCG in at hormonal program for induction/synchronization of estrus and ovulation in 12- 14 months old beef heifers

O presente estudo realizou uma avaliação acerca do efeito do tratamento com eCG e acetato de medroxiprogesterona (MAP) sobre a eficiência reprodutiva em novilhas de corte. Para tanto, em uma primeira etapa (pré-experimento), foram utilizadas 64 novilhas cruzadas (Bos taurus x Bos índicus), de 12 a 14 meses de idade, divididas ao acaso em dois grupos: eCG (n = 32) e controle (n = 32). Na segunda etapa, utilizou-se um número maior de fêmeas (n = 92) da mesma idade e padrão fenotípico, a fim de confirmar os resultados alcançados no grupo eCG. Fêmeas dos grupos-tratamento (eCG e confirmação), no dia zero (início do tratamento), receberam durante sete dias um pessário vaginal contendo 250 mg de MAP, junto a uma injeção intramuscular (IM) de benzoato de estradiol (BE) na dose de 2,5 mg. Nas 24 h antes da retirada do pessário (dia 6), foram aplicados 104 µg de cloprostenol sódico, via submucosa vulvar, e uma injeção IM de 250UI de eCG. Após a retirada dos pessários (dia 7), as fêmeas tiveram seu estro controlado pelo período de 48 h, sendo inseminadas nas 12 h da sua manifestação. As fêmeas que não manifestaram estro nesse período receberam uma injeção IM de 100 µg de GnRH e, após 16-18 h, foram inseminadas sem observação dele (IATF). As fêmeas do grupo-controle receberam o mesmo tratamento hormonal anterior sem o emprego do eCG.(AU)

The present study evaluated the effect of eCG and medroxyprogesterone acetate (MAP) on the reproductive efficiency of beef heifers. Initially, a preliminary experiment comprehending sixty-four 12-14 months old crossbred (Bos taurus x Bos indicus) beef heifers randomly assigned to groups eCG (n = 32) and control (n=32) was performed. In a second moment, ninety-two heifers of same age and fenotipic background were used as a replicate of the first experiment to confirm the preliminary results. Animals from group eCG received an intravaginal sponge pessary containing MAP (250 mg) on day 0 (beginning of treatment) for 7 days, along with an intramuscular (IM) injection of estradiol benzoate (2.5 mg). Twenty-four hours before sponge withdrawal (day 6), cloprostenol sodium (104 µg) was injected in the vulvar submucosa along with an IM injection of eCG (250 IU). After sponge withdrawal on day 7 estrous detection was carried out for forty-eight hours, and animals were inseminated twelve hours after being observed in estrous. Animals which did not manifested estrous received an IM injection of GnRH (100 µg) and were inseminated 16-18 hours later without estrous detection. Animals in the control group were treated according to the same hormonal protocol used for group eCG except for the eCG injection which it was administered.(AU)

Avaliação do emprego do eCG em um programa hormonal de indução/sincronização de estro e ovulação em novilhas de corte entre 12 e 14 meses de idade / Evaluation of the use of eCG in at hormonal program for induction/synchronization of estrus and ovulation in 12- 14 months old beef heifers

O presente estudo realizou uma avaliação acerca do efeito do tratamento com eCG e acetato de medroxiprogesterona (MAP) sobre a eficiência reprodutiva em novilhas de corte. Para tanto, em uma primeira etapa (pré-experimento), foram utilizadas 64 novilhas cruzadas (Bos taurus x Bos índicus), de 12 a 14 meses de idade, divididas ao acaso em dois grupos: eCG (n = 32) e controle (n = 32). Na segunda etapa, utilizou-se um número maior de fêmeas (n = 92) da mesma idade e padrão fenotípico, a fim de confirmar os resultados alcançados no grupo eCG. Fêmeas dos grupos-tratamento (eCG e confirmação), no dia zero (início do tratamento), receberam durante sete dias um pessário vaginal contendo 250 mg de MAP, junto a uma injeção intramuscular (IM) de benzoato de estradiol (BE) na dose de 2,5 mg. Nas 24 h antes da retirada do pessário (dia 6), foram aplicados 104 µg de cloprostenol sódico, via submucosa vulvar, e uma injeção IM de 250UI de eCG. Após a retirada dos pessários (dia 7), as fêmeas tiveram seu estro controlado pelo período de 48 h, sendo inseminadas nas 12 h da sua manifestação. As fêmeas que não manifestaram estro nesse período receberam uma injeção IM de 100 µg de GnRH e, após 16-18 h, foram inseminadas sem observação dele (IATF). As fêmeas do grupo-controle receberam o mesmo tratamento hormonal anterior sem o emprego do eCG.

The present study evaluated the effect of eCG and medroxyprogesterone acetate (MAP) on the reproductive efficiency of beef heifers. Initially, a preliminary experiment comprehending sixty-four 12-14 months old crossbred (Bos taurus x Bos indicus) beef heifers randomly assigned to groups eCG (n = 32) and control (n=32) was performed. In a second moment, ninety-two heifers of same age and fenotipic background were used as a replicate of the first experiment to confirm the preliminary results. Animals from group eCG received an intravaginal sponge pessary containing MAP (250 mg) on day 0 (beginning of treatment) for 7 days, along with an intramuscular (IM) injection of estradiol benzoate (2.5 mg). Twenty-four hours before sponge withdrawal (day 6), cloprostenol sodium (104 µg) was injected in the vulvar submucosa along with an IM injection of eCG (250 IU). After sponge withdrawal on day 7 estrous detection was carried out for forty-eight hours, and animals were inseminated twelve hours after being observed in estrous. Animals which did not manifested estrous received an IM injection of GnRH (100 µg) and were inseminated 16-18 hours later without estrous detection. Animals in the control group were treated according to the same hormonal protocol used for group eCG except for the eCG injection which it was administered.

Control of ovulation in mammals

The ovulatory L H surge induces dramatic molecular and structural changes in follicular environment, culminating with follicle rupture and release of a mature oocyte. This review addresses the pre - LH surge events associated with the ovulatory process, such as dominant fol licle differentiation and acquisition of ovulatory capacity, as well as autocrine and paracrine factors induced after the LH surge. Over the last few years, our research group has focused on studying the contribution of renin - angiotensin system in ovulatio n and the participation of Ang II and Ang - (1 - 7) and their interactions with factors essential for ovulation, which are also discussed.

Control of ovulation in mammals

The ovulatory L H surge induces dramatic molecular and structural changes in follicular environment, culminating with follicle rupture and release of a mature oocyte. This review addresses the pre - LH surge events associated with the ovulatory process, such as dominant fol licle differentiation and acquisition of ovulatory capacity, as well as autocrine and paracrine factors induced after the LH surge. Over the last few years, our research group has focused on studying the contribution of renin - angiotensin system in ovulatio n and the participation of Ang II and Ang - (1 - 7) and their interactions with factors essential for ovulation, which are also discussed.(AU)

Role of angiotensin II on follicle development and ovulation

We will address, in this review, the role of angiotensin II (AngII) on follicular development and ovulation. Over the last few years, our research group has focused on studying the contribution of renin-angiotensin system in antral follicle development and ovulation and a new concept of local regulation has been established using cattle as a model. We previously demonstrated that AT1 and AT2 receptors are expressed in both granulosa and theca cells. The abundance of AT2 mRNA in granulosa cells was higher in healthy compared with atretic follicles, whereas both receptors in theca cells and AT1 in granulosa cells did not change. Granulosa cells cultured with hormones that stimulate estradiol secretion increased AT2 mRNA and protein levels, whereas fibroblast growth factors (FGF-7 and 10) inhibited estradiol secretion and AT2 protein levels. We also found that the concentration of AngII increases in dominant follicle at expected time for follicular deviation. Transvaginal ultrasound has been used for intrafollicular injection to understand the regulation of follicular wave and ovulation. With this in vivo model, we have demonstrated that AngII-receptor blocker inhibits follicular growth and decreases estradiol concentration in follicular fluid and downregulates mRNA expression of genes involved in follicular development. Moreover, intrafollicular injection of AngII or AT2-specific agonist prevented the expected atresia of the second largest follicle, which continued to grow at a rate similar to the dominant follicle for 24 h. These findings have provided evidence that AngII plays an important role in follicle development. In regarding to ovulation, we demonstrated that AngII antagonists block ovulation in cattle when intrafollicularly injected at 0 and 6 h after applying GnRH agonist. Ovulation was also inhibited by AT2- but not by AT1-AngII receptor antagonist. Furthermore, AngII stimulates an enhancement in mRNA abundance of genes involved in ovulation. In addition, AngII stimulates genes involved in extracellular remodeling and follicular wall rupture. In conclusion, our data from in vitro and in vivo studies have demonstrated that AngII plays a pivotal role in the antral follicle development and early mechanism of ovulation via the AT2 receptor subtype in cattle.

Role of angiotensin II on follicle development and ovulation

We will address, in this review, the role of angiotensin II (AngII) on follicular development and ovulation. Over the last few years, our research group has focused on studying the contribution of renin-angiotensin system in antral follicle development and ovulation and a new concept of local regulation has been established using cattle as a model. We previously demonstrated that AT1 and AT2 receptors are expressed in both granulosa and theca cells. The abundance of AT2 mRNA in granulosa cells was higher in healthy compared with atretic follicles, whereas both receptors in theca cells and AT1 in granulosa cells did not change. Granulosa cells cultured with hormones that stimulate estradiol secretion increased AT2 mRNA and protein levels, whereas fibroblast growth factors (FGF-7 and 10) inhibited estradiol secretion and AT2 protein levels. We also found that the concentration of AngII increases in dominant follicle at expected time for follicular deviation. Transvaginal ultrasound has been used for intrafollicular injection to understand the regulation of follicular wave and ovulation. With this in vivo model, we have demonstrated that AngII-receptor blocker inhibits follicular growth and decreases estradiol concentration in follicular fluid and downregulates mRNA expression of genes involved in follicular development. Moreover, intrafollicular injection of AngII or AT2-specific agonist prevented the expected atresia of the second largest follicle, which continued to grow at a rate similar to the dominant follicle for 24 h. These findings have provided evidence that AngII plays an important role in follicle development. In regarding to ovulation, we demonstrated that AngII antagonists block ovulation in cattle when intrafollicularly injected at 0 and 6 h after applying GnRH agonist. Ovulation was also inhibited by AT2- but not by AT1-AngII receptor antagonist. Furthermore, AngII stimulates an enhancement in mRNA abundance of genes involved in ovulation. In addition, AngII stimulates genes involved in extracellular remodeling and follicular wall rupture. In conclusion, our data from in vitro and in vivo studies have demonstrated that AngII plays a pivotal role in the antral follicle development and early mechanism of ovulation via the AT2 receptor subtype in cattle.(AU)

Profile and regulation of annexin II expression during early embryogenesis in cattle / Perfil e regulação da expressão da anexina II durante a embriogênese em bovinos

The presence of annexin II (Ann-II) during the initial stages of bovine embryo development and the regulation of Ann-II expression by retinol and insulin-like growth factor I (IGF-I) were studied. Bovine embryos at different stages of development were produced in vitro on Synthetic Oviductal Fluid (SOF) medium (control group), SOF supplemented with retinol (retinol group; 0.1ng/ml), or IGF-I (IGF-I group; 10ng/ml). The embryos were processed for mRNA extraction, cDNA production and polymerase chain reaction (PCR) using Ann-II-specific oligonucleotides. Ann-II was detected in all stages of early embryo development, except for the 16-cell stage. The blastocyst rates were significantly higher (P<0.05) in the group supplemented with retinol (37.8 percent, 45/119) during in vitro embryo culture (IVC) than in those cultured in SOF (20.5 percent, 24/117) or SOF with IGF-I (25.8 percent, 24/93). Semiquantitative analysis of Ann-II expression in embryos produced in medium supplemented with IGF-I or retinol revealed a lower expression of this gene when compared with embryos cultured in SOF (P<0.05). The Ann-II expression was not different in embryos cultured in the presence of retinol and IGF-I. The presence of retinol increased the production of embryos in vitro by decreasing the expression of Ann-II in early-stage of bovine embryo(AU)

Foram estudadas a presença de anexina II (Ann-II) durante a fase inicial do desenvolvimento embrionário bovino e sua regulação pelo retinol e pelo fator de crescimento semelhante à insulina (IGF-I). Embriões bovinos em diferentes estádios de desenvolvimento foram produzidos in vitro em fluido sintético de oviduto (SOF) sem suplementação (grupo-controle) ou suplementado com retinol (grupo retinol; 0,1ng/ml medium) ou IGF-I (grupo IGF-I; 10 ng/ml de meio). Os embriões foram processados para extração de mRNA, produção de cDNA e posterior análise por reação em cadeia da polimerase (PCR) com oligonucleotídeos específicos para Ann-II. Em todos os estádios de desenvolvimento embrionário, Ann-II foi detectada, exceto no estádio de 16 células. Os índices de blastocisto foram significativamente maiores (P<0,05) no grupo suplementado com retinol (37,8 por cento, 45/119) durante o cultivo in vitro de embriões (PIV) que aqueles obtidos no grupo controle (20,5 por cento, 24/117) ou no IGF-I (25,8 por cento, 24/93). Análise semiquantitativa da expressão de Ann-II em embriões produzidos em meio suplementado com IGF-I ou retinol revelou uma menor expressão desse gene quando comparado com embriões cultivados somente em SOF (P<0,05). A expressão de Ann-II não foi diferente em embriões cultivados na presença de retinol e IGF-I. A presença de retinol aumentou a produção de embriões in vitro, e diminuiu a expressão de Ann-II em estádios iniciais do desenvolvimento embrionário bovino(AU)

Associação entre polimorfismos do gene IGF-IR e características produtivas e reprodutivas em fêmeas bovinas da raça Holandesa / Association between IGF-IR gene polymorphisms and productive and reproductive traits in Holstein cows

Estudou-se a associação entre polimorfismos de conformação de fita simples (SSCP) no gene do receptor do fator-I de crescimento semelhante à insulina (IGF-IR) e idade ao primeiro parto (IPP), intervalo entre partos (IEP), duração da lactação (DL) e produção de leite (PL), em 106 fêmeas puras por cruza da raça Holandesa. A reação em cadeia da polimerase (PCR) com oligonucleotídeos iniciadores específicos gerou um fragmento de 335pb. A freqüência genotípica da população para o polimorfismo foi 82,1% de indivíduos homozigotos para o alelo A e 17,9% de heterozigotos (AB). A freqüência do alelo A foi 0,91 e a do alelo B, 0,09. Não foi encontrada associação entre o polimorfismo estudado e as características IPP, IEP e PL. A característica DL foi positivamente associada (P<0,05) à ausência do alelo B. A lactação dos animais portadores do genótipo AA foi mais longa.(AU)

The association between single-strand conformation polymorphism (SSCP) in the gene of insulin-like growth factor-I receptor (IGF-IR) and age at first calving (AFC), calving interval (CI), lactation length (LL), and milk yield (MY) was studied using 106 graded Holstein females. The polimerase chain reaction (PCR) with specific initiating oligonucleotides, resulted an amplified fragment of 335pb. The population genotypes frequencies were 82.1% and 17.9%, for AA and AB genotypes, respectively. The frequency of A allele was 0.91 and 0.09 of B allele. No association between the identified polymorphism and AFC, CI, and MY was observed. The LL was positively associated (P<0.05) with the absence of B allele. Animals carrying the AA genotype presented a longer lactation period.(AU)

Interação entre células do cumulus e atividade da proteína quinase C em diferentes fases da maturação nuclear de oócitos bovinos / Interaction between cumulus cells and the activity of protein kinase C at different stages of bovine oocyte nuclear maturation

Verificou-se a influência da proteína quinase C (PK-C) no reinício e na progressão da meiose em oócitos bovinos, determinando se as células do cumulus são mediadoras da PK-C na regulação da maturação dos oócitos. Complexos cumulus-oócitos (CCO) e oócitos desnudos (OD), distribuídos aleatoriamente em seis tratamentos (T) com base na presença de um ativador da PK-C (PMA) (T1 e T2), de um forbol éster incapaz de ativar a PK-C (4a-PDD-controle) (T3 e T4) ou de apenas o meio básico (TCM-199-controle) (T5 e T6), foram cultivados por 7, 9, 12, 18 e 22 horas. A percentagem de rompimento da vesícula germinativa no grupo cultivado com PMA foi maior do que nos dois grupos controle, com e sem células do cumulus. O cultivo de CCO e OD por 12 e 18 horas demonstrou que a PK-C influencia a progressão para os estádios de metáfase I (MI) e metáfase II (MII) de maneira dependente das células do cumulus. Nos períodos de 9 e 22 horas, não foi possível observar diferença entre os grupos quanto aos diferentes estádios de maturação. A ativação da PK-C acelera o reinício da meiose independentemente das células somáticas e acelera a progressão até os estádios de MI e MII na dependência das células do cumulus.(AU)

The aim of this study was to evaluate the effect of protein kinase C (PK-C) on the meiotic resumption and progression in bovine oocyte, and to determine if the cumulus cells mediate the PK-C action in the regulation of bovine oocyte nuclear maturation. Cumulus-oocyte complexes (COC) and denuded oocytes (DO), randomly allotted to 6 treatments (T) based on the presence of an activator of PK-C (PMA) (T1 and T2), or a phorbol ester unable to activate PK-C (4aPDD-control) (T3 and T4) or a basic culture medium (T5 and T6), were cultivated for 7, 9, 12, 18 and 22 hours. The percentage of germinal vesicle breakdown (GVBD) was higher when the oocytes were cultured with PMA than in the control groups with and without cumulus cells. However, PK-C was dependent of cumulus cells to affect the progression to the stages of metaphase I (MI) and metaphase II (MII) at 12 and 18 hours of culture. At 9 and 22 hours, no difference among groups was detected. PK-C accelerates the meiotic resumption independently of the somatic cells but depends on cumulus cells for the progression to the stages of MI and MII.(AU)

Efeito da somatotrofina bovina (bST-r), do implante de progestágeno e do desmame por 72 horas na indução do estro e na taxa de prenhez em vacas

This study aimed to assess the efficiency of hormonal treatment in the postpartum fertility of nursing beef cows. Seventy-three cows (Hereford x Nellore) raised extensively, with body condition ranging from 2 to 4 were randomly allotted in three groups. The GSED group, with 25 cows, received an intravaginal device (ID) of 250mg of medroxiprogesteron acetate and 500mg of recombinant bovine somatotropin (bST-r; day 0). After the ID withdrawal (day 7), the cows received 0.5mg of estradiol cipionate; their calves were temporarily weaned for 72 hours. In the SED group, 25 cows received a similar treatment, however, somatotropin was not used. In the control group, 23 cows were temporarily separated from their calves for 72 hours and did not receive any hormonal treatment. The cows were put together with the bulls for 30 days just after ID withdrawal. The cows were weighted and assessed for body condition at day 0 and just after bulls withdrawal. The cows lost weight in an average of 0.648 kg/day, and 56.5%, 33.3%, and 26.1% of estrous rates were observed, respectively for the GSED, SED and control groups (GSED vs. SED, P=0.0001; GSED vs. control, P=0.0007; SED vs. control, P=0.53). The pregnancy rates were 21.7%, 8.3%, and 13.0%, respectively for the GSED, SED and control groups (P=0.16). The results showed that the hormonal program associated with temporary 72-hour calf removal did not increase conception rate of cows that loosed weight from 50 to 70 days postpartum.

Este trabalho teve por objetivo avaliar a eficiência de tratamentos hormonais sobre a fertilidade de vacas de corte no pós-parto com diferentes condições corporais, durante a estação de monta de outono. Setenta e três vacas pluríparas cruzadas (Hereford x Nelore) criadas extensivamente, com condição corporal entre 2 e 4, foram pesadas e distribuídas em três grupos experimentais. O grupo GSED, constituído por 25 vacas, recebeu pessário vaginal (dia 0) contendo 250mg de acetato de medroxiprogesterona e 500mg de somatotropina bovina recombinante (bST-r). Na retirada dos pessários (dia 7), as vacas receberam 0,5mg de cipionato de estradiol e procedeu-se o desmame temporário dos bezerros por 72 horas. No grupo SED, 25 vacas receberam tratamento semelhante ao grupo GSED, porém não receberam bST-r. No grupo-controle, as 23 vacas somente foram separadas dos seus bezerros por 72h. Quando da retirada dos pessários as vacas foram colocadas com touros por 30 dias. Os animais foram pesados e avaliados quanto à condição corporal no início do experimento e na retirada dos touros (dia 37). Foi constatada perda média de peso de 0,648 kg/dia e os percentuais de estro foram de 26,1%, 33,3% e 56,5%, respectivamente, para os grupos controle, SED e GSED. O diagnóstico de gestação, realizado pela palpação retal 60 dias após a retirada dos touros, indicou percentuais de prenhez de 13,0%, 8,3% e 21,7%, respectivamente, para os grupos controle, SED e GSED (P=0,16), demonstrando que os programas hormonais adotados não foram eficientes no incremento das taxas de prenhez de vacas que perdiam peso entre 50 e 70 dias após o parto.

Fator de crescimento derivado das plaquetas, retinol e insulina na regulação da maturação nuclear de oócitos bovinos e suas conseqüências no desenvolvimento embrionário / Platelet-derived growth factor, retinol and insulin in the regulation of bovine oocyte nuclear maturation and their consequent effect in the embryonic development

O objetivo desse trabalho foi verificar as ações do fator de crescimento derivado das plaquetas (PDGF;P), da insulina (I), do retinol (R) e de suas associações(PI, PIR, IR e PR) na maturação nuclear (MN)de oócitos bovinos e suas consequências no desenvolvimento embrionário (DE). O meio básico para maturação dos oócitos nos diferentes tratamentos foi o TCM-199 modificado acrescido de PVA (controle). No DE, foram utilizados os grupos R, PIR, IR, um controle negativo (PVA) e um controle positivo, contendo soro fetal bovino e gonadotrofinas (SFBHOR). Os fatores P, I, R e suas associações não aceleraram a MN em 7h mas sim após 18h (P<0,001), com exceção dos tratamentos R e PR, nos quais as percentagens de metáfase II foram, respectivamente, de 4,7(porcento) e 8,3(porcento), similares à obtida no grupo-controle (0,0(porcento)). Considerando um nível de significância de P<0,0001 em comparação ao grupo-controle, os maiores índices de matáfase II foram obtidos na presença das associações IR (19,0(porcento)) e PIR (21,3(porcento)). No DE, R (18,3(porcento)), PIR (13,9(porcento)) e IR (10,6(porcento)) incrementaram os índices de clivagem, comparados ao PVA (0,0(porcento);P<0,001), porém não atingiram os índices do grupo SFBHOR (53,8(porcento);P<0,001). Conclui-se que insulina e PDGF aceleram a MN e suas ações são potencializadas pelo retinol. Os índices de clivagem de oócitos maturados na presença de R, IR e PIR são superiores aos do PVA, mas significativamente inferiores aos maturados em SFBHOR (AU)

The aim of the present study was to determine the effect of platelet-derived growth factor (PDGF; P), insulin (I) retinol, (R) and their interactions (PI, PIR, IR and PR) on oocyte nuclear maturation (NM) and, consequent, embryonic development (ED). The basic medium for oocyte maturation in the treatments was the modified TCM-199, supplemented with PVA (control). To study the embryonic development, the oocytes were divided in three treatments, R, PIR e IR, a negative (PVA) and a positive control group (containing calf fetal serum and gonadotrophic hormones; FCSHOR). The PDGF, insulin, retinol and their interactions did not change the kinetic of the NM, in seven hours of culture (P=0.4492) but it changed after 18 hours of maturation (P<0.001) except in the treatments R and PR (P<0.001), in which the percentages of metaphase II were, respectively, 4.7% and 8.3%. These results were similar to the control group (0.0%). Considering a significant level of P<0.0001 in comparison to the control group, the higher rates of metaphase II were obtained in the presence of IR (19.0%) and PIR (21.3%). The higher rates of MII were observed when the oocytes were matured in the presence of insulin and retinol. In the embryonic development, R (18.3%), PIR (13.9%) and IR (10.6%) increased the rate of cleavage when compared to PVA group (0.0%; P<0.001). However, the oocytes were not competent enough to reach the rate obtained in the FCSHOR group (53.8%; P<0.001). In conclusion, insulin and PDGF accelerate NM and their effects are enhanced by retinol. In the embryonic development, oocytes matured in the presence of either R, IR or PIR have higher cleavage rate than PVA group but lower than those matured in the FCSHOR group. (AU)