Chemotactic behavior of Campylobacter fetus subspecies towards cervical mucus, bovine placenta and selected substances and ion

Abstract The chemotaxis of C. fetus subsp. venerealis and C. fetus subsp. fetus was determined in the presence of bovine cervical mucus and bovine placental extract. Some reported substances and ion in those materials, such amino acids, ferrous iron, hormones, sugars and organic acids were also investigated. Bovine cervical mucus, bovine placenta extracts and some substances and ion of these materials namely L-fucose, L- aspartate, L-glutamate, L-serine, ferrous iron, fumarate, pyruvate and succinate were chemoattractants. The chemottraction was significantly larger in higher concentrations of the tested substances and ion and significant differences among tested strains were also observed. Meso-erythritol and hormones bovine placental lactogen, 17β-estradiol, and progesterone did not elicit chemotactical response. In conclusion, this chemotactic behavior may guide the C. fetus navigation in the bovine host's genital tract and be an important cofactor of tissue tropism for this bacterium.

Chemotactic behavior of Campylobacter fetus subspecies towards cervical mucus, bovine placenta and selected substances and ion

The chemotaxis of C. fetus subsp. venerealis and C. fetus subsp. fetus was determined in the presence of bovine cervical mucus and bovine placental extract. Some reported substances and ion in those materials, such amino acids, ferrous iron, hormones, sugars and organic acids were also investigated. Bovine cervical mucus, bovine placenta extracts and some substances and ion of these materials namely L–fucose, Laspartate, L–glutamate, L–serine, ferrous iron, fumarate, pyruvate and succinate were chemoattractants. The chemottraction was significantly larger in higher concentrations of the tested substances and ion and significant differences among tested strains were also observed. Meso-erythritol and hormones bovine placental lactogen, 17β-estradiol, and progesterone did not elicit chemotactical response. In conclusion, this chemotactic behavior may guide the C. fetus navigation in the bovine host's genital tract and be an important cofactor of tissue tropism for this bacterium.(AU)

Evolution of placentation in cattle and antelope

Bovids have enjoyed great evolutionary success as evidenced by the large number of extant species. Several important domestic animals are from this family. They derive from both subfamilies: cattle and their kin belong to Bovinae and sheep and goats to Antilopinae. The premise of this review, therefore, is that evolution of reproduction and placentation is best understood in a context that includes antelope-like bovines and antelopes. Many key features of placentation, including hormone secretion, had evolved before bovids emerged as a distinct group. Variation nevertheless occurs. Most striking is the difference in fusion of the binucleate trophoblast cell with uterine epithelium that yields a transient trinucleate cell in bovines and many antelopes, but a more persistent syncytium in wildebeest, sheep and goat. There is considerable variation in placentome number and villus branching within the placentome. Many antelopes have right-sided implantation in a bicornuate uterus whilst others have a uterus duplex. Finally, there has been continued evolution of placental hormones with tandem duplication of PAG genes in cattle, differences in glycosylation of placental lactogen and the emergence of placental growth hormone in sheep and goats. The selection pressures driving this evolution are unknown though maternal-fetal competition for nutrients is an attractive hypothesis.

Evolution of placentation in cattle and antelope

Bovids have enjoyed great evolutionary success as evidenced by the large number of extant species. Several important domestic animals are from this family. They derive from both subfamilies: cattle and their kin belong to Bovinae and sheep and goats to Antilopinae. The premise of this review, therefore, is that evolution of reproduction and placentation is best understood in a context that includes antelope-like bovines and antelopes. Many key features of placentation, including hormone secretion, had evolved before bovids emerged as a distinct group. Variation nevertheless occurs. Most striking is the difference in fusion of the binucleate trophoblast cell with uterine epithelium that yields a transient trinucleate cell in bovines and many antelopes, but a more persistent syncytium in wildebeest, sheep and goat. There is considerable variation in placentome number and villus branching within the placentome. Many antelopes have right-sided implantation in a bicornuate uterus whilst others have a uterus duplex. Finally, there has been continued evolution of placental hormones with tandem duplication of PAG genes in cattle, differences in glycosylation of placental lactogen and the emergence of placental growth hormone in sheep and goats. The selection pressures driving this evolution are unknown though maternal-fetal competition for nutrients is an attractive hypothesis.(AU)

Placental contribution to the endocrinology of gestation and parturition

In addition to many other functions, the placenta is a source of a vast number of autocrine, paracrine and endocrine factors. However, the spectrum of placental regulatory factors, their concentrations, gestational profiles and roles may differ considerably even between phylogenetically closely related species. Depending on the species, placental regulatory factors of a broad range of molecule classes have been found including (glyco- )proteins, peptides, steroids and prostaglandins. Local placental regulatory factors are especially important for the dialogue between the fetal and the maternal compartment immediately at the feto-maternal borderline and for the control of growth, differentiation and functions of the placenta itself. Moreover, placental hormones in a proper sense may also have effects in more remote targets within the maternal compartment, serving functions such as pregnancy-specific adaptations of maternal circulation, provision of hemotrophe to the fetus or the development and function of the mammary gland. Functions of placental hormones in the fetus proper are less clear but may be especially important before the establishment of a functional fetal endocrine system and near term within the highly species-specific networks of signals preparing and initiating parturition. This review takes a comparative view on the situation in different domestic animals focusing on ruminants and on placental hormones occurring at significant concentrations in the maternal circulation.

Placental contribution to the endocrinology of gestation and parturition

In addition to many other functions, the placenta is a source of a vast number of autocrine, paracrine and endocrine factors. However, the spectrum of placental regulatory factors, their concentrations, gestational profiles and roles may differ considerably even between phylogenetically closely related species. Depending on the species, placental regulatory factors of a broad range of molecule classes have been found including (glyco- )proteins, peptides, steroids and prostaglandins. Local placental regulatory factors are especially important for the dialogue between the fetal and the maternal compartment immediately at the feto-maternal borderline and for the control of growth, differentiation and functions of the placenta itself. Moreover, placental hormones in a proper sense may also have effects in more remote targets within the maternal compartment, serving functions such as pregnancy-specific adaptations of maternal circulation, provision of hemotrophe to the fetus or the development and function of the mammary gland. Functions of placental hormones in the fetus proper are less clear but may be especially important before the establishment of a functional fetal endocrine system and near term within the highly species-specific networks of signals preparing and initiating parturition. This review takes a comparative view on the situation in different domestic animals focusing on ruminants and on placental hormones occurring at significant concentrations in the maternal circulation.(AU)

Hiperplasia fibroepitelial mamária felina (HFMF): novas perspectivas de regulação endócrina / Feline mammary fibroepithelial hyperplasia (FMFH): new perspectives of endocrine control

O controle da mamogênese, que inicia na fase embrionária e continua ao longo das várias etapas reprodutivas da fêmea, ainda não foi totalmente elucidado. Pesquisas nessa área geralmente são conduzidas com espécies produtoras de leite para fins comerciais, mas podem auxiliar nas investigações sobre a patogenia da hiperplasia fibroepitelial mamária felina (HFMF). Caracterizada como um fenômeno de crescimento anabólico que ocorre somente em algumas gatas, com proliferação celular nas estruturas alveolares, tanto no epitélio luminal quanto no mioepitélio, a HFMF pode exacerbar ao ponto de romper a pele e expor o parênquima mamário. Do ponto de vista endócrino, observa-se o envolvimento da progesterona, tanto endógena quanto exógena, aliada a uma sinergia com Hormônio do Crescimento (GH) e Fator de crescimento semelhante a insulina-1 (IGF-1). Todavia, a interação com outros hormônios envolvidos na mamogênese ainda é um campo aberto à pesquisa. Objetiva-se com esta revisão discorrer sobre os hormônios e fatores de crescimento celular relacionados à mamogênese e ocorrência de HFMF.(AU)

The control of mamogenesis, which starts on embryonic phase and continues throughout many female reproductive phases, was not fully understood. Researches on this field are commonly performed with dairy species, but support investigations focused on Feline mammary fibroepithelial hyperplasia (FMFH) pathogenesis. Characterized as a phenomenon of anabolic growth that occurs in few female cats, with alveolar proliferation in both luminal and myoepithelial cells, the FMFH can exacerbated and expose mammary parenchyma due to skin disruption. Endocrinologically it is observed participation of endogenous and exogenous progesterone, associated to synergism with Growth hormone (GH) and Insulin like growth factor-1 (IGF-1). However, interaction with other hormones enrolled on mammogenesis remains an open field to research. The goal of this review is to discuss about hormones and cellular growth factors related to mammogenesis and FMFH occurrence.(AU)

Hiperplasia fibroepitelial mamária felina (HFMF): novas perspectivas de regulação endócrina / Feline mammary fibroepithelial hyperplasia (FMFH): new perspectives of endocrine control

O controle da mamogênese, que inicia na fase embrionária e continua ao longo das várias etapas reprodutivas da fêmea, ainda não foi totalmente elucidado. Pesquisas nessa área geralmente são conduzidas com espécies produtoras de leite para fins comerciais, mas podem auxiliar nas investigações sobre a patogenia da hiperplasia fibroepitelial mamária felina (HFMF). Caracterizada como um fenômeno de crescimento anabólico que ocorre somente em algumas gatas, com proliferação celular nas estruturas alveolares, tanto no epitélio luminal quanto no mioepitélio, a HFMF pode exacerbar ao ponto de romper a pele e expor o parênquima mamário. Do ponto de vista endócrino, observa-se o envolvimento da progesterona, tanto endógena quanto exógena, aliada a uma sinergia com Hormônio do Crescimento (GH) e Fator de crescimento semelhante a insulina-1 (IGF-1). Todavia, a interação com outros hormônios envolvidos na mamogênese ainda é um campo aberto à pesquisa. Objetiva-se com esta revisão discorrer sobre os hormônios e fatores de crescimento celular relacionados à mamogênese e ocorrência de HFMF.

The control of mamogenesis, which starts on embryonic phase and continues throughout many female reproductive phases, was not fully understood. Researches on this field are commonly performed with dairy species, but support investigations focused on Feline mammary fibroepithelial hyperplasia (FMFH) pathogenesis. Characterized as a phenomenon of anabolic growth that occurs in few female cats, with alveolar proliferation in both luminal and myoepithelial cells, the FMFH can exacerbated and expose mammary parenchyma due to skin disruption. Endocrinologically it is observed participation of endogenous and exogenous progesterone, associated to synergism with Growth hormone (GH) and Insulin like growth factor-1 (IGF-1). However, interaction with other hormones enrolled on mammogenesis remains an open field to research. The goal of this review is to discuss about hormones and cellular growth factors related to mammogenesis and FMFH occurrence.

Paraoxonase activity in the serum of peripartum dairy cows with different placental lactogen concentrations / Atividade sérica de Paraoxanase durante o periparto de vacas leiteiras com diferentes concentrações de Lactogênio Placentário

The action of the bovine placental lactogen (bPL) hormone on maternal metabolism is still poorly known. Some markers, such as the acute phase protein paraoxonase (PON1), are used as indicators of liver function and help to determine the metabolic condition during the transition period in dairy cows. The aim of this study was to evaluate the activity of paraoxonase (PON1) in the serum of peripartum dairy cows with different levels of bPL. Based on the plasma bPL concentration, 18 cows were divided equally into three groups: LOW ( < 2,68 ng bPL mL-1), MEDIUM (2,682,80 ng bPL mL-1), and HIGH ( > 2,80 ng bPL mL-1). The experiment was conducted between 21 days prepartum and 28 days postpartum. Serum samples were collected during the experiment for the determination of bPL concentrations and PON1 activity. The bPL concentration was significantly different between the experimental groups (P ≤ 0,0001) and the days of serum collection (P ≤ 0,0001). In the prepartum dairy cows, the PON1 levels were different between the groups (P ≤ 0,05) and the days of serum collection (P ≤ 0,05). Cows with high bPL concentration had lower serum PON1 activity (P ≤ 0,05), while cows with low hormone levels had higher enzyme activity (P ≤ 0,05). In the postpartum period, there was a significant difference between the days of serum collection (P ≤ 0,0001) and the interaction between groups and collections(P ≤ 0,01). The group with high concentrations of bPL had lower levels of PON1 (P ≤ 0,01), while thegroup with low bPL maintained higher concentrations of PON1 (P ≤ 0,01). It was concluded that thecows with higher concentrations of bPL in the prepartum period present a reduction in the serum activityof the PON1 enzyme during the peripartum period.(AU)

A ação do hormônio Lactogênio Placentário Bovino (bLP) no metabolismo materno ainda é poucoconhecida. Alguns marcadores, como a proteína de fase aguda Paraoxanase (PON1), são utilizadoscomo indicadores da função hepática auxiliando na determinação da condição metabólica no período de transição em vacas leiteiras. O objetivo deste trabalho foi avaliar a atividade sérica de PON1 duranteo periparto em vacas leiteiras com diferentes níveis de bLP. As vacas foram divididas em três gruposde acordo com as concentrações plasmáticas de bLP, em que BAIXO < 2,68 ng mL-1 (n=6), MÉDIO> 2,68 ng mL-1 e < 2,80 ng mL-1 (n=6) e ALTO > 2,80 ng mL-1 (n=6). O período experimental ocorreuentre os 21 dias pré-parto e 28 dias pós-parto. Amostras de soro foram coletadas para a determinaçãodas concentrações de bLP e atividade sérica de PON1. Houve diferença entre os três grupos (P ≤ 0,0001)de acordo com as concentrações de bLP, assim como entre os dias coletados (P ≤ 0,0001). No pré-parto,os níveis de PON1 apresentaram diferença entre grupos (P ≤ 0,05) e coletas (P ≤ 0,05). Vacas com altaconcentração de bLP apresentaram menor atividade sérica de PON1 (P ≤ 0,05), enquanto vacas combaixos níveis do hormônio obtiveram maior atividade da enzima (P ≤ 0,05). No pós-parto não houvediferença entre grupos (P ≥ 0,10), houve diferença entre os dias coletados (P ≤ 0,0001) e interação entregrupos e coletas (P ≤ 0,01). O grupo com altas concentrações de bLP apresentou menores níveis dePON1 (P ≤ 0,01), enquanto vacas do grupo com baixo bLP mantiveram maiores concentrações de PON1(P ≤ 0,01). Conclui-se que, vacas com maiores concentrações de bLP no período pré-parto apresentamredução na atividade sérica da enzima PON1 durante o período periparto.(AU)

Modulation of uterine function by endocrine and paracri ne factors in ruminants

Uterine adenogenesis in the neonate is critical as uterine glands are essential for pregnancy in adult ruminants. The uterus is stimulated by estrogens (E2) and progesterone (P4) that prepare it to respond to biochemical signals from the conceptus (embryo/fetus and placenta). Interferon ta u (IFNT) is responsible for pregnancy recognition and modification of uterine gene expression including sensitivity to placental lactogen and placental growth hormone that stimulate development and gene expression by epithelial cells of uterine glands. P4 is permissive for most actions of IFNT. Novel genes are expressed by uterine luminal and superficial glandular epithelia in response to P4 and IFNT as those cells are in direct contact with conceptus trophectoderm. But, uterine glandular epithelium and stromal cells respond to P4 and IFNT by expressing classical interferon stimulat ed genes. Uterine receptivity to implantation requires loss of expression of receptors for P4 and E2 by uterine epithelia. P4 stimulates P4 receptor-positive st romal cells to express fibroblast growth factor 10 (FGF10) and hepatocyte growth factor (HGF) that act via their re spective receptors on uterine epithelia and trophectoderm to regulate cellular functions and gene expression. FGF10 and IFNT are hypothesized to activate complementary cell signaling pathways that modulate expression of genes for implantation, modify phenotype of uterine stromal cells, silence expression of genes for P4 and E2 receptors, signal pregnancy recognition, suppress genes for immune recognition, alter membrane permeability to enhance conceptus-maternal exchange of factors, increase endometrial vascularity, and activate genes for transport of nutrients into the uterine lumen. Those actions are essential for a successful outcome of pregnancy.

Modulation of uterine function by endocrine and paracri ne factors in ruminants

Uterine adenogenesis in the neonate is critical as uterine glands are essential for pregnancy in adult ruminants. The uterus is stimulated by estrogens (E2) and progesterone (P4) that prepare it to respond to biochemical signals from the conceptus (embryo/fetus and placenta). Interferon ta u (IFNT) is responsible for pregnancy recognition and modification of uterine gene expression including sensitivity to placental lactogen and placental growth hormone that stimulate development and gene expression by epithelial cells of uterine glands. P4 is permissive for most actions of IFNT. Novel genes are expressed by uterine luminal and superficial glandular epithelia in response to P4 and IFNT as those cells are in direct contact with conceptus trophectoderm. But, uterine glandular epithelium and stromal cells respond to P4 and IFNT by expressing classical interferon stimulat ed genes. Uterine receptivity to implantation requires loss of expression of receptors for P4 and E2 by uterine epithelia. P4 stimulates P4 receptor-positive st romal cells to express fibroblast growth factor 10 (FGF10) and hepatocyte growth factor (HGF) that act via their re spective receptors on uterine epithelia and trophectoderm to regulate cellular functions and gene expression. FGF10 and IFNT are hypothesized to activate complementary cell signaling pathways that modulate expression of genes for implantation, modify phenotype of uterine stromal cells, silence expression of genes for P4 and E2 receptors, signal pregnancy recognition, suppress genes for immune recognition, alter membrane permeability to enhance conceptus-maternal exchange of factors, increase endometrial vascularity, and activate genes for transport of nutrients into the uterine lumen. Those actions are essential for a successful outcome of pregnancy.(AU)

The placenta of bovine clones

Background: Since the first success in sheep, the production of viable cloned offspring by somatic cell nuclear transfer (SCNT) in various mammals has increased significantly. The incidence of pregnancy failure and fetal death, however, is still very high, whatever the species, and impairs the commercial development of this technology, even in the bovine species where the success rates are highest compared to other species. Review: In cattle, most gestation losses are initially due to abnormal implantation and poor placental development leading to fetal demise during the early post-implantation period (30 to 70 days of pregnancy). Thereafter, in continuing pregnancies, losses usually occur in the last third of gestation and affect about 25% of the on-going pregnancies, with very large differences according to phenotype. These are currently referred to as the Large Offspring Syndrome (LOS), Large Placenta Syndrome or Abnormal Offspring Syndrome. In all cases, the placenta appears to be central to the onset of the pathology, with placentomegaly and hydrallantois being the most common features. Clinically, transabdominal ultrasound monitoring of fetal and placental development as well as the measurement of maternal plasma concentrations of pregnancy associated glycoproteins (PAG) are recommended in order to monitor the pregnancies. Humane termination of the pregnancies by Caesarian section or slaughtering of the affected animals is recommended when the pathology onset is diagnosed more than 2 weeks prior to term. Underlying mechanisms include abnormal placental vascularization, which is present early in SCNT placental development. Enzymatic response to oxidative stress is also modified. In the first trimester, several genes expressed in the trophoblast have been found to be differentially expressed between SCNT and control conceptuses, including placental lactogen (PL), the PAG, prolactin related protein-1 (PRP-1) and Dickkopf-1(DKK-1), to name a few. All these proteins are expressed in the Binucleate cells (BNC) of the trophoblast and thus, indicate that BNC function may be affected in SCNT from very early in gestation, thereby compromising placental development. Later in pregnancy, it has been shown that transplacental exchanges are disturbed, in particular those related to glucose metabolism. Moreover, endocrine function is altered compared to controls, with decreased estrogen secretion and modifications in PAG secretion, resulting in largely elevated maternal plasma concentrations. Gene expression patterns are affected, with most prominent functional effects involving cell cycle, cell signaling pathways, molecular transport, DNA replication, recombination and repair. Most of the affected genes are downregulated. Finally, many of the pathologies reported with SCNT pregnancies resemble abnormalities reported with either mutations or deletions of imprinted genes or dysregulation of imprinted gene expression, and the expression of several imprinted genes have been shown to be abnormal in SCNT placenta. Conclusions: In conclusion, pregnancy failure after SCNT is due to multiple factors affecting, implantation, placental development, morphology, vascularization, responses to oxidative stress and the epigenetic control of gene expression. If abnormal nuclear reprogramming may induce long term effects in bovine SCNT, these effects may also be due to fetal programming due to abnormal placental function and perturbed fetal development.

Quimiotaxia de Campylobacter fetus por substâncias do útero bovino gestante

Campylobacter fetus é um patógeno que coloniza o trato genital e placenta bovina, no entanto, os mecanismos envolvidos no tropismo tecidual deste microrganismo são desconhecidos. Vários mecanismos são importantes para a afinidade tecidual de Campylobacter spp., incluindo a quimiotaxia. Neste estudo foi avaliada a atividade quimiotática de ácidos orgânicos, aminoácidos, carboidratos e íon inorgânico, componentes do muco uterino, nas concentrações entre 0.005M a 1M e hormônios produzidos pela placenta bovina nas concentrações entre 0.005 ng/mL a 500 ng/mL para duas amostras de C. fetus subsp. venerealis e quatro amostras de C. fetus subsp. fetus. Meso-eritritol, 17-estradiol e lactogênio placentário bovino não apresentaram atividade quimiotática, nas concentrações testadas, para as amostras avaliadas, enquanto deoxicolato de sódio foi quimiorepelente. L-fucose, sulfato de ferro ferroso, fumarato de sódio, piruvato de sódio, succinato de sódio, L-aspartato, L-glutamato e L-serina foram quimioatraentes para as amostras avaliadas de C. fetus subsp. venerealis e C. fetus subsp. fetus. Progesterona foi quimioatraente, em concentrações acima de 50 ng/mL, para duas amostras de C. fetus subsp. fetus. Assim, este estudo demonstrou que substâncias químicas presentes no útero e placenta bovina atraem C. fetus subsp. venerealis e C. fetus subsp. fetus e que duas amostras de C. fetus subsp. fetus foram atraídas por altas concentrações de progesterona.

Campylobacter fetus is a pathogen that colonizes the bovine genital tract and placenta, however, the mechanisms responsible for the tissue tropism of this microorganism are unknown. Several mechanisms are important for tissue affinity of Campylobacter spp., including chemotaxis. In this study, the chemotactic activity of organic acids, amino acids, carbohydrates and inorganic ion, common uterine mucous components, in concentrations from 0.005M to 1M, and hormones produced by the bovine placenta, in concentrations of 0.005 ng/mL to 500 ng/mL, was evaluated for two strains of C. fetus subsp. venerealis and four strains of C. fetus subsp. fetus. Meso-erythritol, 17-estradiol and bovine placental lactogen presented no chemotactic activity in the concentrations tested, while sodium deoxycholate was chemorepellent for all strains tested. L-fucose, iron ferrous sulfate, sodium fumarate, sodium pyruvate, sodium succinate, L-aspartate, L-glutamate and L-serine were chemoattractants for the evaluated C. fetus subsp. venerealis and C. fetus subsp. fetus strains. Progesterone was chemoattractant, in concentrations above 50 ng/mL, for two strains of C. fetus subsp. fetus. Thus, this study showed that chemical substances present in the bovine uterus and placenta attract C. fetus subsp. venerealis and C. fetus subsp. fetus and that two strains of C. fetus subsp. fetus were attracted by high concentrations of progesterone

Interação celular na placenta de gestações de bovinos clonados com especial ênfase às integrinas / Cell-cell interactions in the bovine placenta from pregnancies with cloned fetuses with special respect to integrins and their receptors

Integrinas são glicoproteínas transmembrânicas envolvidas na adesão célula-célula e célula-proteina da matriz extracellular (ECM) que participam da migração e fusão de células trofoblásticas gigantes (TGC) com células do epitélio materno no placentoma bovino e interagem com inibidores teciduais de metalloproteinases (TIMPs), considerados importantes reguladores das metaloproteinases de matriz (MMPs), que são reconhecidas como passo limitande para ação de enzimas que atuam no remodelamento da ECM na implantação e na gestação. Uma vez que as integrinas são consideradas responsáveis pela manutenção da arquitetura tecidual e que as MMPs podem regular a degradação e a reconstituição da ECM necessária para a invasão do trofoblasto, temos como objetivo verificar uma possível relação das integrinas com alterações placentárias comumente observadas em gestações de animais clonados (SCNT). Para avaliar a funcionalidade placentária, a expressão do lactogênio placentário (PL) e do antígeno Ki-67 foram também verificadas. Placentomas bovinos foram coletados e divididos em 3 grupos: 1) início (n = 6) e 2) final (n = 3) da gestação de animais derivados de monta natural (n=9) e final da gestação de animais clonados (n=8), clones machos (n=4) e clones fêmeas (n=4). As proteínas foram localizadas por imuno-histoquímica indireta, exceto as subunidades de integrina α6, β1, αV e β3 que foram localizadas por imunofluorescência. A especificidade dos anticorpos e expressão protéica foi confirmada por Western blot e a expressão do RNAm foi avaliada por meio de RT-PCR e PCR em tempo real quantitativo. As células positivas para a laminina, TIMP-2, Ki-67 e lactogênio placentário (PL) foram quantificadas e para comparação entre os grupos. A proteína das subunidades α6 do receptor de integrina e β1 foi observada nos animais clonados e não clonados nos estromas materno e fetal e porção basal dos epitélios materno e fetal e foi co-localizada com a laminina. A proteína das subunidades αV e β3 do receptor de integrina foi observado nos estromas materno e fetal e foi co-localizado com a fibronectina. O TIMP-2 foi exclusivamente e especificamente localizado nas TGCs no início e final da gestação de animais clonados e não clonados, mas não houve diferença significativa em sua expressão. O PL apresentou a mesma localização do TIMP-2 em todos os grupos analisados. Houve diferença significativa (p<0,05) entre 270d e clones ao se comprar a expressão relativa do RNAm da subunidade de integrina β1 e do lactogênio placentário e ao se comparar a expressão protéica por western blot da laminina e do TIMP-2. Foi possível observar diferença significativa (p<0,05) entre o grupo controle e o grupo de animais clonados ao se quantificar o número de TGCs positivas para a laminina e para o lactogênio placentário. Apesar de algumas diferenças terem sido observadas na expressão de integrinas e de proteínas da ECM, sugere-se que a interação das integrinas com seus ligantes da ECM a termo não é um determinante da sobrevivência neonatal de bovinos clonados. Contudo, outros estudos são necessários para esclarecer se estas proteínas representam elementos chave na placentação de bovinos clonados em início de gestação. As diferenças observadas na expressão de integrinas e principalmente do PL entre clones fêmeas e machos sugerem possível influência de genes associados ao cromossomo sexual ou imprinting

Integrins are transmembrane glycoproteins involved in cell-cell and cell-extracellular matrix (ECM) adhesion and signal transduction. They participate in the migration and fusion of trophoblast giant cells (TGC) with uterine epithelial cells in bovine placentomes, and interact with tissue inhibitors of metalloproteinases (TIMPs), considered important regulators of matrix metalloproteinases (MMPs). MMPs are rate-limiting enzymes degrading ECM proteins during tissue remodeling around embryo implantation, pregnancy and parturition. Since integrins are considered to be responsible for the maintenance of the architecture within tissues and MMPs may also regulate degradation and reconstitution of ECM required for trophoblast invasion, we aimed to verify a potential relation of integrins with common placental alterations observed in cloned animals (SCNT). To verify the placental functionality, expression of placental lactogen (PL) and Ki-67 antigen was also assessed. Bovine placentomes were collected and divided into 3 groups: 1) early and 2) late gestation derived from natural mating (n=9) and 3) late gestational bovine clones (n=8), also divided into male clones (n=4) and female clones (n=4). All proteins were localized by indirect immunohistochemistry except for integrin subunits α6, β1, αV and β3 that were shown by immunofluorescence. The antibody specificity and protein expression were confirmed by Western blot. Expression of mRNA was evaluated using RT-PCR and quantitative Real Time PCR. Positive cells for laminin, TIMP-2, Ki-67 and placental lactogen were quantified for comparisons among groups. The protein of integrin receptor subunits α6 and β1 was observed in both cloned and non-cloned animals in the fetal and maternal stroma and at the basal membrane of maternal and fetal epithelial cells, co-localized with laminin and with collagen IV. The integrin receptor subunits αV and β3 were observed in the fetal and maternal stroma, co-localized with fibronectin. TIMP-2 was specifically and exclusively localized in TGC in the early and term gestation in both cloned and non-cloned animals. PL has shown the same localization of TIMP-2 in all analyzed samples. Statistically significant differences (p<0,05) between term gestation of non-cloned and cloned animals were observed comparing the mRNA expression of integrin β1 subunit and placental lactogen and the protein expression of laminin and TIMP-2. There were also statistically significant differences (p<0,05) between non-cloned and cloned animals in the number of laminin and placental lactogen positive cells. Despite some differences in integrin and ECM proteins expression, our results suggest that the interaction between integrins and their ECM ligands in term gestation is not a determinant for the survival rate of newborn cloned calves. However further studies are necessary to elucidate if integrins represent key elements in the early placentation of SCNT bovines. The differences found in the integrin and principally in the placental lactogen expression between female and male cloned calves suggests the influence of sex-chromosome associated genes or imprinting

Paraoxonase activity in the serum of peripartum dairy cows with different placental lactogen concentrations / Atividade sérica de Paraoxanase durante o periparto de vacas leiteiras com diferentes concentrações de Lactogênio Placentário

The action of the bovine placental lactogen (bPL) hormone on maternal metabolism is still poorly known. Some markers, such as the acute phase protein paraoxonase (PON1), are used as indicators of liver function and help to determine the metabolic condition during the transition period in dairy cows. The aim of this study was to evaluate the activity of paraoxonase (PON1) in the serum of peripartum dairy cows with different levels of bPL. Based on the plasma bPL concentration, 18 cows were divided equally into three groups: LOW ( 2,80 ng bPL mL-1). The experiment was conducted between 21 days prepartum and 28 days postpartum. Serum samples were collected during the experiment for the determination of bPL concentrations and PON1 activity. The bPL concentration was significantly different between the experimental groups (P ≤ 0,0001) and the days of serum collection (P ≤ 0,0001). In the prepartum dairy cows, the PON1 levels were different between the groups (P ≤ 0,05) and the days of serum collection (P ≤ 0,05). Cows with high bPL concentration had lower serum PON1 activity (P ≤ 0,05), while cows with low hormone levels had higher enzyme activity (P ≤ 0,05). In the postpartum period, there was a significant difference between the days of serum collection (P ≤ 0,0001) and the interaction between groups and collections(P ≤ 0,01). The group with high concentrations of bPL had lower levels of PON1 (P ≤ 0,01), while thegroup with low bPL maintained higher concentrations of PON1 (P ≤ 0,01). It was concluded that thecows with higher concentrations of bPL in the prepartum period present a reduction in the serum activityof the PON1 enzyme during the peripartum period.

A ação do hormônio Lactogênio Placentário Bovino (bLP) no metabolismo materno ainda é poucoconhecida. Alguns marcadores, como a proteína de fase aguda Paraoxanase (PON1), são utilizadoscomo indicadores da função hepática auxiliando na determinação da condição metabólica no período de transição em vacas leiteiras. O objetivo deste trabalho foi avaliar a atividade sérica de PON1 duranteo periparto em vacas leiteiras com diferentes níveis de bLP. As vacas foram divididas em três gruposde acordo com as concentrações plasmáticas de bLP, em que BAIXO 2,68 ng mL-1 e 2,80 ng mL-1 (n=6). O período experimental ocorreuentre os 21 dias pré-parto e 28 dias pós-parto. Amostras de soro foram coletadas para a determinaçãodas concentrações de bLP e atividade sérica de PON1. Houve diferença entre os três grupos (P ≤ 0,0001)de acordo com as concentrações de bLP, assim como entre os dias coletados (P ≤ 0,0001). No pré-parto,os níveis de PON1 apresentaram diferença entre grupos (P ≤ 0,05) e coletas (P ≤ 0,05). Vacas com altaconcentração de bLP apresentaram menor atividade sérica de PON1 (P ≤ 0,05), enquanto vacas combaixos níveis do hormônio obtiveram maior atividade da enzima (P ≤ 0,05). No pós-parto não houvediferença entre grupos (P ≥ 0,10), houve diferença entre os dias coletados (P ≤ 0,0001) e interação entregrupos e coletas (P ≤ 0,01). O grupo com altas concentrações de bLP apresentou menores níveis dePON1 (P ≤ 0,01), enquanto vacas do grupo com baixo bLP mantiveram maiores concentrações de PON1(P ≤ 0,01). Conclui-se que, vacas com maiores concentrações de bLP no período pré-parto apresentamredução na atividade sérica da enzima PON1 durante o período periparto.