The in vitro effects of n-3 fatty acids on immune response regulation of bovine ex vivo endometrial explants / Os efeitos in vitro dos ácidos graxos n-3 na regulação da resposta imune de explantes endometriais bovinos ex vivo

Fatty acids are considered metabolic intermediaries, although new facts indicate they also work as signaling molecules with different roles in the immune response. Based on that, in this study, we investigated the anti-inflammatory effects of n-3 polyunsaturated fatty acids (PUFAs) as eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and α-linolenic acid (LNA) in ex vivo bovine endometrial explants. For this, two groups were formed: (1) LPS-challenged and (2) control, both to evaluate the accumulation of proinflammatory cytokines as interleukin 1ß (IL1B) and interleukin 6 (IL6). To develop the study, bovine female reproductive tracts from non-pregnant Angus heifers without evidence of reproductive diseases were selected. Endometrial explants were processed and treated for 24 h with EPA, DHA, and LNA in five different concentrations (0µM, 50µM, 100 µM, 200µM and 400 µM) and then, challenged with LPS for 24 h. Supernatants were collected to evaluate the concentration of IL1B and IL6 by ELISA. Explants treated with EPA from control groups reduced the concentrations of ILB (200µM) and IL6 (400 µM), and IL6 (50 µM; 100 µM) from the LPS-challenged group. DHA decreased the accumulation of IL1B and IL6 at 200 µM on explants from the LPS-challenged group, and 200 µM reduced IL6 from the control group. In contrast, explants treated with LNA only reduced the accumulation of IL1B to 400µM (from both groups). In conclusion, the EPA acid is the best anti-inflammatory option to decrease the concentration of both pro-inflammatory cytokines (IL1B and IL6) from LPS-challenged and control groups in bovine endometrial explants; while LNA evidence to be the last option to promote an anti-inflammatory response.(AU)

Os ácidos graxos são considerados intermediários metabólicos, embora novos fatos indiquem que eles também atuem como moléculas sinalizadoras com diferentes papéis na resposta imune. Dessa forma, este estudo investigou os efeitos anti-inflamatórios de ácidos graxos poliinsaturados n-3 (PUFAs) como ácido eicosapentaenóico (EPA), ácido docosahexaenóico (DHA) e ácido α-linolênico (LNA) em explantes endometriais ex vivo de bovinos. Para tal, o experimento foi divido em dois grupos: (1) Desafiado-LPS e (2) Controle, para que então pudesse avaliar o acúmulo de citocinas pró-inflamatórias como interleucina 1ß (IL1B) e interleucina 6 (IL6). Foram selecionados tratos reprodutivos de fêmeas bovinas de novilhas Angus não prenhes sem evidência de doenças reprodutivas. Explantes endometriais foram processados e tratados por 24h com EPA, DHA e LNA em cinco concentrações diferentes (0µM, 50µM, 100 µM, 200µM e 400 µM) e, em seguida, desafiados com LPS por mais 24h. Os sobrenadantes foram colhidos para avaliar a concentração de IL1B e IL6 pelo teste de ELISA. Os explantes tratados com EPA dos grupos de controle reduziram as concentrações de ILB (200 µM) e IL6 (400 µM) e no grupo desafiado com LPS houve redução das concentrações de IL6 (50 µM; 100 µM). Nos explantes do grupo desafiado com LPS, o DHA diminuiu o acúmulo de IL1B e IL6 nas concentrações de 200 µM, e no grupo controle reduziu IL6 nas concentrações de 200 µM, enquanto os explantes tratados com LNA reduziram apenas o acúmulo de IL1B a 400 µM (de ambos os grupos). Em conclusão, o ácido EPA provou ser a melhor opção anti-inflamatória para diminuir a concentração de ambas as citocinas pró-inflamatórias (IL1B e IL6) de grupos desafiados com LPS e controle em explantes endometriais bovinos; enquanto o LNA evidencia ser a opção menos viável para promover uma resposta anti-inflamatória.(AU)

Ovários policisticos epidermóides e hipoplasia uterina em uma vaca bovina / Epidermoid polycystic ovaries and uterine hypoplasia in a cow

Background: Ovarian cysts originate from mature follicles that do not ovulate within the predicted time according to the oestrous cycle. Dermoid cysts are structures lined by keratinized stratified squamous epithelium, without adjacent epidermal structures, filled by keratinic debris and amorphous proteinaceus material. They are small, located below the cortex, near the hilus. There is controversy regarding the teratogenicity of the dermoid cyst. Some authors use the nomenclature of benign cystic ovarian teratoma, others, reports that although the fibrous wall and histological features are similar to teratomas, it is not associated with this germ cell-derived neoplasia. Case: Ovaries and uterus were received from a female, adult bovine, Nelore, from a slaughterhouse. The left ovary measured 6.5x5.0x3.9 cm and weighed 80 g; the right ovary measured 5.7x3.7x3.0 cm and weighed 60 g. Grossly, the ovaries were similar, presenting floating consistency, multilobulated and pointed aspect, and at the cut surface, there was extravasation of mucopurulent content of whitish colour. Regarding to the uterus, macroscopically, the uterine horns were infantile and decrease in the diameter. Fragments of the tissues were collected and fixed in 10% buffered formalin for histopathological examination and the staining of the slides was done with hematoxylin and eosin. Microscopically, in the ovaries, it was observed rare vestigial elements of ovary identifying an atresic follicle. Polycystic formation with cysts exhibiting partial or total coating of a keratinized squamous epithelium was also observed. In the lumen abundant keratinous material was present. The uterine microscopy revealed compact endometrium with areas variably infiltrated by lymphocytes, plasmocytes and occasional neutrophils. Areas devoid of endometrial glands were observed

Ovários policisticos epidermóides e hipoplasia uterina em uma vaca bovina / Epidermoid polycystic ovaries and uterine hypoplasia in a cow

Background: Ovarian cysts originate from mature follicles that do not ovulate within the predicted time according to the oestrous cycle. Dermoid cysts are structures lined by keratinized stratified squamous epithelium, without adjacent epidermal structures, filled by keratinic debris and amorphous proteinaceus material. They are small, located below the cortex, near the hilus. There is controversy regarding the teratogenicity of the dermoid cyst. Some authors use the nomenclature of benign cystic ovarian teratoma, others, reports that although the fibrous wall and histological features are similar to teratomas, it is not associated with this germ cell-derived neoplasia. Case: Ovaries and uterus were received from a female, adult bovine, Nelore, from a slaughterhouse. The left ovary measured 6.5x5.0x3.9 cm and weighed 80 g; the right ovary measured 5.7x3.7x3.0 cm and weighed 60 g. Grossly, the ovaries were similar, presenting floating consistency, multilobulated and pointed aspect, and at the cut surface, there was extravasation of mucopurulent content of whitish colour. Regarding to the uterus, macroscopically, the uterine horns were infantile and decrease in the diameter. Fragments of the tissues were collected and fixed in 10% buffered formalin for histopathological examination and the staining of the slides was done with hematoxylin and eosin. Microscopically, in the ovaries, it was observed rare vestigial elements of ovary identifying an atresic follicle. Polycystic formation with cysts exhibiting partial or total coating of a keratinized squamous epithelium was also observed. In the lumen abundant keratinous material was present. The uterine microscopy revealed compact endometrium with areas variably infiltrated by lymphocytes, plasmocytes and occasional neutrophils. Areas devoid of endometrial glands were observed…(AU)

Aspects of embryo-maternal communication in establishment of pregnancy in cattle

Establishment of pregnancy in mammals requires reciprocal molecular communication between the conceptus and endometrium that modifies the endometrial transcriptome and uterine luminal milieu to support pregnancy. Due to the small size of the early embryo and elongating conceptus relative to the volume of the uterine lumen, collection of endometrium adjacent to the developing conceptus is difficult following conventional uterine flushing methods in cattle. Use of endometrial explants in culture can overcome this challenge and reveal information about the dialogue between the developing embryo and the uterus. The aim of this short review is to summarize some of our recent findings in relation to embryo maternal interaction during bovine pregnancy establishment and to put them in the wider context of fertility in cattle.

Aspects of embryo-maternal communication in establishment of pregnancy in cattle

Establishment of pregnancy in mammals requires reciprocal molecular communication between the conceptus and endometrium that modifies the endometrial transcriptome and uterine luminal milieu to support pregnancy. Due to the small size of the early embryo and elongating conceptus relative to the volume of the uterine lumen, collection of endometrium adjacent to the developing conceptus is difficult following conventional uterine flushing methods in cattle. Use of endometrial explants in culture can overcome this challenge and reveal information about the dialogue between the developing embryo and the uterus. The aim of this short review is to summarize some of our recent findings in relation to embryo maternal interaction during bovine pregnancy establishment and to put them in the wider context of fertility in cattle.(AU)

Parthenogenetic bovine embryos secrete type I interferon capable of stimulating ISG15 in luteal cell culture

Interferon tau (IFNT) is the pregnancy recognition signal in ruminants and is secreted by trophoblast cells. Paracrine action in the endometrium is well established by inhibiting luteolytic pulses of prostaglandin F2 alpha. Recently, endocrine action was documented in the corpus luteum, blood cell and liver. It was hypothesized that conditioned medium (CM) obtained from days 7, 9 and 12 parthenogenetic embryos alters luteal cell gene expression. The aim was to establish a bovine mixed luteal cell culture to evaluate cellular response associated to interferon stimulated genes, steroidogenesis and apoptosis. Conditioned medium was obtained from Days 7, 9 and 12 parthenogenetic (PA) embryos culture. Moreover, antiviral assay was performed on CM from Days 7, 9 and 12 to verify Type I interferon activity. Luteal cell culture was validated by steroidogenic and apoptotic genes (CYP11A1, HSD3B1, BAX, BCL2, AKT and XIAP mRNA expression), and concentration of progesterone as endpoint. Luteal cell culture was treated with interferon alpha (IFNA) and CM from parthenogenetic embryos. Antiviral assay revealed Type I interferon activity on CM from embryos increasing on Days 9 and 12. ISG15 mRNA was greater in the mixed luteal cells culture treated with 1, 10 and 100ng/ml of interferon alpha (IFNA) and also on Days 7, 9 and 12 CM treatments. Concentration of progesterone was not altered in luteal cell culture regardless of treatments. Steroidogenic and apoptotic genes were similar among groups in luteal cell culture treated with different doses of IFNA or CM from PA embryos. In conclusion, parthenogenetic embryo-derived CM has antiviral activity, luteal cell culture respond to Type I interferon by expressing IGS15. These data indicate this model can be used for IFNT endocrine signaling studies.

Parthenogenetic bovine embryos secrete type I interferon capable of stimulating ISG15 in luteal cell culture

Interferon tau (IFNT) is the pregnancy recognition signal in ruminants and is secreted by trophoblast cells. Paracrine action in the endometrium is well established by inhibiting luteolytic pulses of prostaglandin F2 alpha. Recently, endocrine action was documented in the corpus luteum, blood cell and liver. It was hypothesized that conditioned medium (CM) obtained from days 7, 9 and 12 parthenogenetic embryos alters luteal cell gene expression. The aim was to establish a bovine mixed luteal cell culture to evaluate cellular response associated to interferon stimulated genes, steroidogenesis and apoptosis. Conditioned medium was obtained from Days 7, 9 and 12 parthenogenetic (PA) embryos culture. Moreover, antiviral assay was performed on CM from Days 7, 9 and 12 to verify Type I interferon activity. Luteal cell culture was validated by steroidogenic and apoptotic genes (CYP11A1, HSD3B1, BAX, BCL2, AKT and XIAP mRNA expression), and concentration of progesterone as endpoint. Luteal cell culture was treated with interferon alpha (IFNA) and CM from parthenogenetic embryos. Antiviral assay revealed Type I interferon activity on CM from embryos increasing on Days 9 and 12. ISG15 mRNA was greater in the mixed luteal cells culture treated with 1, 10 and 100ng/ml of interferon alpha (IFNA) and also on Days 7, 9 and 12 CM treatments. Concentration of progesterone was not altered in luteal cell culture regardless of treatments. Steroidogenic and apoptotic genes were similar among groups in luteal cell culture treated with different doses of IFNA or CM from PA embryos. In conclusion, parthenogenetic embryo-derived CM has antiviral activity, luteal cell culture respond to Type I interferon by expressing IGS15. These data indicate this model can be used for IFNT endocrine signaling studies.(AU)

Influência das endometrites nas estruturas ovarianas e qualidade oocitária em tratos reprodutivos post-mortem de vacas / Influence of endometritis on ovarian structures and oocyte quality in post mortem reproductive tract of cows

Objetivou-se no presente estudo estimar o percentual de endometrites clínicas (EC) e subclínicas (ES) em tratos reprodutivos post-mortem de fêmeas bovinas, verificando sua influência sobre as estruturas ovarianas, alterações presentes no ovário, número e qualidade de oócitos recuperados. Foram coletados e avaliados 171 tratos reprodutivos em matadouro frigorífico. As endometrites foram diagnosticadas por meio de citologia endometrial e pela presença e características das secreções uterinas. Os diagnósticos foram confirmados pela análise histopatológica, sendo avaliada a existência de infiltrados de células inflamatórias no endométrio. Os ovários foram avaliados macroscopicamente quanto à estrutura e alterações ovarianas. A qualidade oocitária foi avaliada de acordo com o número de camadas de células do cumulus e aspecto do citoplasma, sendo assim, classificados como: grau I (GI), grau II (GII), grau III (GIII) e grau IV (GIV). Dentre as peças avaliadas, 8,20% (n = 14) apresentaram EC e 4,10% (n = 7) foram diagnosticadas com ES. Observou-se que a presença de endometrites não exerceu efeito sobre as estruturas e alterações ovarianas, assim como não alterou o número de oócitos recuperados. Em relação ao grau de qualidade oocitária, foi encontrado variação entre os diferentes graus de qualidade dentro dos grupos, no entanto, não houve correlação aparente com a presença de infecção uterina.(AU)

The objective of this study was to estimate the percentage of clinical (CE) and subclinical (SE) endometritis in post mortem bovine reproductive tracts, analyzing their influence on ovarian structures, changes in the ovary, and number and quality of oocytes collected. Endometritis were diagnosed by endometrial cytology, and by the presence and characteristics of uterine secretions. The diagnosis was confirmed through histopathological analysis, by evaluating the presence of infiltrated inflammatory cells in the endometrium. The ovaries were macroscopically evaluated for presence of physiological structures and alterations. The oocyte quality evaluation was performed according to the number of cumulus cells layers and cytoplasmic aspect, being classified as: grade I (GI), grade II (GII), grade III (GIII) and grade IV (GIV). Among the uterus evaluated, 8,20% (n = 14) of the animals presented EC, and 4,10% (n = 7) were diagnosed with ES. It was observed that the presence of endometritis had no effect on ovarian structures and alterations, nor did it influence the number of oocytes collected. Regarding the degree of oocyte quality, a variation was found between the different grades of quality within the groups, however, there was no apparent correlation with the presence of uterine infection.(AU)

Influência das endometrites nas estruturas ovarianas e qualidade oocitária em tratos reprodutivos post-mortem de vacas / Influence of endometritis on ovarian structures and oocyte quality in post mortem reproductive tract of cows

Objetivou-se no presente estudo estimar o percentual de endometrites clínicas (EC) e subclínicas (ES) em tratos reprodutivos post-mortem de fêmeas bovinas, verificando sua influência sobre as estruturas ovarianas, alterações presentes no ovário, número e qualidade de oócitos recuperados. Foram coletados e avaliados 171 tratos reprodutivos em matadouro frigorífico. As endometrites foram diagnosticadas por meio de citologia endometrial e pela presença e características das secreções uterinas. Os diagnósticos foram confirmados pela análise histopatológica, sendo avaliada a existência de infiltrados de células inflamatórias no endométrio. Os ovários foram avaliados macroscopicamente quanto à estrutura e alterações ovarianas. A qualidade oocitária foi avaliada de acordo com o número de camadas de células do cumulus e aspecto do citoplasma, sendo assim, classificados como: grau I (GI), grau II (GII), grau III (GIII) e grau IV (GIV). Dentre as peças avaliadas, 8,20% (n = 14) apresentaram EC e 4,10% (n = 7) foram diagnosticadas com ES. Observou-se que a presença de endometrites não exerceu efeito sobre as estruturas e alterações ovarianas, assim como não alterou o número de oócitos recuperados. Em relação ao grau de qualidade oocitária, foi encontrado variação entre os diferentes graus de qualidade dentro dos grupos, no entanto, não houve correlação aparente com a presença de infecção uterina.

The objective of this study was to estimate the percentage of clinical (CE) and subclinical (SE) endometritis in post mortem bovine reproductive tracts, analyzing their influence on ovarian structures, changes in the ovary, and number and quality of oocytes collected. Endometritis were diagnosed by endometrial cytology, and by the presence and characteristics of uterine secretions. The diagnosis was confirmed through histopathological analysis, by evaluating the presence of infiltrated inflammatory cells in the endometrium. The ovaries were macroscopically evaluated for presence of physiological structures and alterations. The oocyte quality evaluation was performed according to the number of cumulus cells layers and cytoplasmic aspect, being classified as: grade I (GI), grade II (GII), grade III (GIII) and grade IV (GIV). Among the uterus evaluated, 8,20% (n = 14) of the animals presented EC, and 4,10% (n = 7) were diagnosed with ES. It was observed that the presence of endometritis had no effect on ovarian structures and alterations, nor did it influence the number of oocytes collected. Regarding the degree of oocyte quality, a variation was found between the different grades of quality within the groups, however, there was no apparent correlation with the presence of uterine infection.

Regressão espontânea do corpo lúteo em bovinos - revisão / Spontaneous corpus luteum regression in cattle: review

O conhecimento do processo de luteólise dentro da fisiologia reprodutiva se torna importante para que ocorra a compreensão do reconhecimento materno da gestação e seja possível formular estratégias anti-luteolíticas que visem manter esta fase conferindo aumentos na eficiência reprodutiva e produtiva. Durante o processo de luteólise, o tecido luteal sofre mudanças bruscas na capacidade esteroidogênica, vascularização e remodelamento, resultando em substituição da glândula por tecido conjuntivo, sendo a PGF2α o principal determinante na indução espontânea da luteólise em bovinos. Simultaneamente, ocorre um íntimo processo entre as relações anatômicas que compreende o ovário, útero e suas vascularizações. A exposição do útero à interação coordenada entre P4, E2 e ocitocina aliada à expressão de seus receptores são essenciais para a secreção pulsátil de PGF2α pelo endométrio a qual induzirá a luteólise. Nesta regressão do corpo lúteo (CL), estão envolvidos ainda os hormônios (LH, prolactina, cortisol) e várias substâncias vasoativas (óxido nítrico, VEGF, EG-VEGF, bFGF, END-1, AngII). A concentração de PGF2α pode ser representada pela mensuração sanguínea da concentração de seu metabólito 13,14-dihidro-15-ceto- PGF2α (PGFM) sendo a pulsatilidade da secreção de PGF2α durante os períodos pré-luteolítico, luteolítico e pós-luteolítico mensurável pelo aumento na concentração de PGFM. Neste artigo de revisão, será possível compreender e discutir os principais aspectos endócrinos e moleculares que ocorrem com as mudanças morfofuncionais no CL durante o processo de luteólise fisiológica em fêmeas bovinas.

The knowledge of the luteolysis process within reproductive physiology becomes important to occur the understanding of maternal recognition of gestation being possible to formulate anti-luteolytic strategies aimed at maintaining this phase conferring increases in reproductive and productive efficiency. During the luteolysis process, luteal tissue undergoes abrupt changes in steroidogenic capacity, vascularization and remodeling, resulting in replacement of the gland by connective tissue, and PGF2α is the main determinant in the spontaneous induction of luteolysis in cattle. Simultaneously, an intimate process occurs between the anatomical relationships that comprise the ovary, uterus and its vascularizations. The exposure of the uterus to the coordinated interaction between P4, E2 and oxytocin, together with the expression of its receptors, are essential for the pulsating secretion of PGF2α by the endometrium, which will induce luteolysis. In this regression of the corpus luteum (CL), hormones (LH, prolactin, cortisol) and various vasoactive substances (nitric oxide, VEGF, EG-VEGF, bFGF, END-1, AngII) are also involved. The concentration of PGF2α can be represented by the blood measurement of the concentration of its metabolite 13,14-dihydro-15-keto-PGF2α (PGFM),being the pulsatility of PGF2α secretion during the pre-luteolytic, luteolytic and post-luteolytic measured by the increased concentration of PGFM. In this review article, it will be possible to understand and discuss the main endocrine and molecular aspects that occur with the morphofunctional changes in CL during the physiological luteolysis process in bovine females.

Regressão espontânea do corpo lúteo em bovinos - revisão / Spontaneous corpus luteum regression in cattle: review

O conhecimento do processo de luteólise dentro da fisiologia reprodutiva se torna importante para que ocorra a compreensão do reconhecimento materno da gestação e seja possível formular estratégias anti-luteolíticas que visem manter esta fase conferindo aumentos na eficiência reprodutiva e produtiva. Durante o processo de luteólise, o tecido luteal sofre mudanças bruscas na capacidade esteroidogênica, vascularização e remodelamento, resultando em substituição da glândula por tecido conjuntivo, sendo a PGF2α o principal determinante na indução espontânea da luteólise em bovinos. Simultaneamente, ocorre um íntimo processo entre as relações anatômicas que compreende o ovário, útero e suas vascularizações. A exposição do útero à interação coordenada entre P4, E2 e ocitocina aliada à expressão de seus receptores são essenciais para a secreção pulsátil de PGF2α pelo endométrio a qual induzirá a luteólise. Nesta regressão do corpo lúteo (CL), estão envolvidos ainda os hormônios (LH, prolactina, cortisol) e várias substâncias vasoativas (óxido nítrico, VEGF, EG-VEGF, bFGF, END-1, AngII). A concentração de PGF2α pode ser representada pela mensuração sanguínea da concentração de seu metabólito 13,14-dihidro-15-ceto- PGF2α (PGFM) sendo a pulsatilidade da secreção de PGF2α durante os períodos pré-luteolítico, luteolítico e pós-luteolítico mensurável pelo aumento na concentração de PGFM. Neste artigo de revisão, será possível compreender e discutir os principais aspectos endócrinos e moleculares que ocorrem com as mudanças morfofuncionais no CL durante o processo de luteólise fisiológica em fêmeas bovinas.(AU)

The knowledge of the luteolysis process within reproductive physiology becomes important to occur the understanding of maternal recognition of gestation being possible to formulate anti-luteolytic strategies aimed at maintaining this phase conferring increases in reproductive and productive efficiency. During the luteolysis process, luteal tissue undergoes abrupt changes in steroidogenic capacity, vascularization and remodeling, resulting in replacement of the gland by connective tissue, and PGF2α is the main determinant in the spontaneous induction of luteolysis in cattle. Simultaneously, an intimate process occurs between the anatomical relationships that comprise the ovary, uterus and its vascularizations. The exposure of the uterus to the coordinated interaction between P4, E2 and oxytocin, together with the expression of its receptors, are essential for the pulsating secretion of PGF2α by the endometrium, which will induce luteolysis. In this regression of the corpus luteum (CL), hormones (LH, prolactin, cortisol) and various vasoactive substances (nitric oxide, VEGF, EG-VEGF, bFGF, END-1, AngII) are also involved. The concentration of PGF2α can be represented by the blood measurement of the concentration of its metabolite 13,14-dihydro-15-keto-PGF2α (PGFM),being the pulsatility of PGF2α secretion during the pre-luteolytic, luteolytic and post-luteolytic measured by the increased concentration of PGFM. In this review article, it will be possible to understand and discuss the main endocrine and molecular aspects that occur with the morphofunctional changes in CL during the physiological luteolysis process in bovine females.(AU)

Basic and practical aspects of pregnancy establishment in cattle

Bovine embryos are increasingly produced using reproductive technologies, e.g. ovum pick-up (OPU), in vitro embryo production (IVP) and embryo transfer (ET). Such in vitro manipulated embryos are known to deviate in several aspects compared to in vivo derived embryos. Pregnancy establishment in cattle involves timed biological events including fine-tuned communication, initiated and carried out by both the embryo and the endometrium. This stimulates research to increase the understanding of events and interactions taking place in the uterus after embryo transfer, both from a biological and systems biology point of view. This review will focus on the biological events taking place during early embryonic development, implantation and beginning of placentation, with focus on transfer of in vitro produced embryos, including a systems biology approach for selection of superior embryo recipients.

Basic and practical aspects of pregnancy establishment in cattle

Bovine embryos are increasingly produced using reproductive technologies, e.g. ovum pick-up (OPU), in vitro embryo production (IVP) and embryo transfer (ET). Such in vitro manipulated embryos are known to deviate in several aspects compared to in vivo derived embryos. Pregnancy establishment in cattle involves timed biological events including fine-tuned communication, initiated and carried out by both the embryo and the endometrium. This stimulates research to increase the understanding of events and interactions taking place in the uterus after embryo transfer, both from a biological and systems biology point of view. This review will focus on the biological events taking place during early embryonic development, implantation and beginning of placentation, with focus on transfer of in vitro produced embryos, including a systems biology approach for selection of superior embryo recipients.(AU)

Extracellular vesicles secreted by bovine embryos produced in vivo and in vitro: miRNAs content and molecular effects in endometrial and luteal tissues

BRIDI, A. Vesículas extracelulares secretadas por embriões bovinos produzidos in vivo e in vitro: conteúdo de miRNAs e os efeitos moleculares sobre o endométrio e o corpo lúteo. 254 f. Tese (Doutorado) Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo, Pirassununga-SP, 2021. Perfis metabólicos, padrões de expressão gênica, desenvolvimento embrionário, capacidade de estabelecer e manter gestações precoces são diferenças encontradas quando embriões bovinos produzidos in vivo e in vitro são comparados. Para que a gestação tenha sucesso é necessária uma perfeita comunicação entre o embrião e o organismo materno. Vesículas extracelulares pequenas (sEVs) fazem parte dessa comunicação e carregam moléculas bioativas, como miRNAs, que podem atuar em células-alvo dentro do sistema reprodutivo bovino. Com isso, nossa hipótese é que embriões bovinos produzidos in vivo e in vitro secretam sEVs contendo diferentes miRNAs que podem modular diferencialmente o padrão de expressão gênica endometrial e o perfil de miRNAs do corpo lúteo. Para isso, primeiramente, investigamos o conteúdo de miRNA em blastocistos eclodidos produzidos in vivo ou in vitro e, em sEVs secretadas por eles. Blastocistos eclodidos produzidos in vivo ou in vitro, no dia 9 de desenvolvimento, possuem diferentes perfis de miRNA. EVs pequenas secretadas por eles, do dia 7 até o dia 9 de desenvolvimento, contém miRNAs diferentes. Esses miRNAs diferentemente expressos são preditos por regularem vias envolvidas no desenvolvimento embrionário inicial e na receptividade endometrial. Por meio disso, as primeiras interações entre o embrião e o organismo materno podem ser modificadas, e isso pode afetar o sucesso da gestação. Em segundo lugar, investigamos as alterações no transcriptoma global do endométrio após o co-cultivo de explantes endometriais com um blastocisto produzido in vivo ou in vitro no dia 7 do desenvolvimento. Genes diferentemente expressos foram identificados no endométrio e estão associados com a presença e origem do embrião. Além disso, sEVs presentes nos meios condicionados (C.M.) por essas células embrionárias e endometriais contém diferentes miRNAs, que são preditos por modularem a via de sinalização da oxitocina. Finalmente, para compreender melhor se a comunicação entre o embrião, o endométrio e o corpo lúteo pode ser intermediada por sEVs, explantes luteais foram tratados com sEVs presentes nos C.M. por explantes endometriais cultivados sozinhos ou com um blastocisto bovino produzido in vivo ou in vitro no dia 7 do desenvolvimento. O perfil de miRNAs foi modificado nos explantes luteais tratados com essas sEVs e, nós podemos observar um efeito da presença e origem dos embriões nessas alterações. Juntos, esses resultados sugerem que sEVs intermedeiam a comunicação inicial entre o embrião, endométrio e o corpo lúteo, contribuindo para manter a viabilidade e funcionalidade luteal. Entretanto, futuros experimentos precisam ser realizados para avaliar efeitos biológicos específicos desses miRNAs carreados por essas sEVs no endométrio e no corpo lúteo. Além disso, a origem do embrião (in vivo ou in vitro) modifica as interações entre ele, o endométrio e o corpo lúteo, sugerindo fortemente que esses embriões tenham necessidades distintas para se desenvolverem.

BRIDI, A., Extracellular vesicles secreted by bovine embryos produced in vivo and in vitro: miRNAs content and molecular effects in endometrial and luteal tissues. 2021. 254 f. Tese (Doutorado) Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo, Pirassununga-SP, 2021. Metabolic profiles, gene expression patterns, embryonic development, and the ability to establish and sustain early pregnancies are differences founded when in vivo- and in vitro- produced bovine embryos are compared. To the establishment of a successful pregnancy the perfect embryo- maternal communication is needed. Small extracellular vesicles (sEVs) are part of this crosstalk and carry bioactive molecules such as miRNAs that can act on target bovine cells within thereproductive system. Thereby, our hypothesis is that in vivo and in vitro bovine embryos secrete sEVs containing different miRNAs that can differentially modulate endometrial gene expression pattern and miRNAs profile in the corpus luteum (CL). To address this hypothesis, firstly, we investigated the miRNA content of in vivo and in vitro hatched blastocysts and in sEVs secreted by them. Day 9 in vivo or in vitro hatched blastocysts have distinct miRNA profiles. Small EVs secreted by them from day 7 up to day 9 of development contain different miRNAs. These miRNAs differently expressed are predicted to regulate pathways involved with early embryonic development and endometrial receptivity. Thereby, early embryo-maternal interactions can be modified and consequently can affect pregnancy success. Secondly, we investigated changes in the endometrial global transcriptome after the co-culture of endometrial explants with a day 7 in vivo or in vitro blastocysts. Differently expressed genes were identified in the endometrium and are associated with the embryo's presence or origin. Moreover, sEVs present in the conditioned media (C.M.) by these embryonic and endometrial cells contain different miRNAs, which are predicted to modulate the oxytocin signaling pathway. Finally, to understand if the communication among embryo, endometrium, and corpus luteum can be mediated by sEVs, luteal explants were treated with sEVs from C.M. by endometrial explants alone or cultured with a day 7 in vivo or in vitro blastocysts. MicroRNAs profile was modified in luteal explants treated with these sEVs, and we can observe an effect of the embryo presence and origin in these alterations. Together these results suggest that sEVs mediate early embryo-endometrium-corpus luteum communication, contributing to maintaining luteal viability and functionality. However, further experiments are needed to evaluate specific biological effects of these miRNAs carried by sEVs in the endometrium and in the corpus luteum. Moreover, the embryo origin (in vivo and in vitro) modify the interactions among embryo-endometrium-corpus luteum, suggesting strongly that these embryos have distinct needed to develop.

FATORES QUE AFETAM A SÍNTESE DE MEDIADORES INFLAMATÓRIOS DA RESPOSTA IMUNE INATA ENDOMETRIAL DE BOVINOS

A ocorrência de doenças uterinas no pós-parto de vacas leiteiras é elevada e impacta diretamente nos índices reprodutivos dos animais. Assim sendo, este trabalho teve como objetivo avaliar a expressão gênica de mediadores pró-inflamatórios aos 14 e 35 dias em lactação (DEL) no endométrio de vacas mestiças leiteiras com e sem endometrite subclínica (ES). Os animais foram avaliados semanalmente aos sete, 14, 28 e 35 DEL e realizados os exames físico e do sistema reprodutor. Aos 14 e 35 DEL foram coletadas amostras endometriais por meio de biópsia uterina. As amostras foram processadas e analisadas pela técnica de qRT-PCR, para avaliação da expressão gênica das citocinas pró-inflamatórias IL1, IL6, IL8 e CCL5. Além disso, aos 35 DEL foram coletados swabs por meio da técnica de escova endometrial (cytobrush) para citologia endometrial e diagnóstico da ES. A expressão gênica da IL1 foi maior no grupo sem doença comparado ao grupo ES aos 14 DEL (p=0,0009). Houve diferença na expressão gênica da IL8 entre os grupos (p<0,001) aos 14 DEL e em relação ao momento de coleta (p = 0,0011), com maior expressão aos 14 DEL quando comparado aos 35 DEL. Não foram observadas diferenças na expressão gênica da IL6 e CCL5 entre os grupos com e sem ES e os momentos avaliados (14 x 35 DEL). Concluiu-se que as vacas mestiças leiteiras sem doenças uterinas pós-parto apresentam resposta imune mais robusta aos 14 DEL, com maior expressão das citocinas pró-inflamatórias IL1 e IL8 quando comparadas às vacas com ES, sendo a análise da expressão gênica destas citocinas uma ferramenta potencial para o diagnóstico precoce da ES. Além disso, tendo em vista que a kisspeptina é um neuropeptídeo conhecido por sua potente ação estimulatória do hormônio liberador de gonadotrofina (GNRH), responsável por regular centralmente a função ovariana e o ciclo estral. Além do seu papel fundamental na regulação do eixo reprodutivo, a kisspeptina é capaz de modular a função das células do sistema imune, com possível regulação na interação destes dois sistemas. O presente estudo também objetivou avaliar o papel modulatório da kisspeptina na resposta imune inata endometrial de fêmeas bovinas. Para tanto, foram realizados cinco experimentos e utilizado o total de 24 tratos reprodutivos de fêmeas bovinas nulíparas, coletados em abatedouro comercial, para processamento através da técnica de cultivo de endométrio ex vivo. Os tratamentos utilizados no cultivo dos explantes endometriais de regiões caruncular e intercaruncular consistiram de kisspeptina (Kp 10-5 M; Kp 10-6 M; Kp 10-7 M), antagonista p234 (p234 10-5 M; p234 10-7 M; p234 10-9 M), progesterona (5ng/mL) e dexametasona (5ng/mL), desafiados ou não com 1g/mL lipopolissacarídeo (LPS), nos tempos de 12 e 24 horas de cultivo. Foram avaliadas a expressão gênica das citocinas IL6, IL1 e a concentração das citocinas IL6, IL1 e IL8, pelas técnicas de qRT-PCR e ELISA, respectivamente. Não houve diferença na expressão gênica das citocinas IL1 e IL6 e na produção de IL6, IL1 e IL8 em nenhum dos tratamentos realizados com diferentes concentrações de kisspeptina. Concluiu-se que a kisspeptina não apresentou efeito modulatório sobre a resposta imune inata do endométrio bovino quando se utilizou o modelo de cultivo endometrial ex vivo.

Due to the high occurrence of uterine diseases in the postpartum period of dairy cows and directly impacting the reproductive indices of animals, this study also aimed to evaluate the gene expression of proinflammatory mediators in the endometrium of crossbred dairy cows with and without SE, at 14 and 35 days in milk (DIM). The physical and reproductive system evaluations of the animals were performed weekly at 7, 14, 28 and 35 DIM. At 14 and 35 DIM, endometrial samples were collected by uterine biopsy. The samples were analyzed by the qRT-PCR technique, to evaluate the gene expression of the proinflammatory cytokines IL1, IL6, IL8 and CCL5. In addition, it was collected endometrial swabs using the endometrial brush technique (cytobrush) for endometrial cytology and diagnosis of SE. The IL1 gene expression to the group without SE was higher than the SE group at 14 DIM (p = 0.0009). There was a difference in the IL8 gene expression at 14 DIM between groups (p < 0.001) and in relation to the collection time, with greater expression at 14 DIM when compared to 35 DIM (p = 0.0011). There were no differences in the IL6 and CCL5 gene expression between cows with and without SE and the collection time. In conclusion, the crossbred dairy cows without uterine diseases have a more robust immune response at 14 DIM, with greater expression of the proinflammatory cytokines IL1 and IL8 when compared to the cows with SE, being the analysis of the gene expression of these cytokines a potential tool for the early diagnosis of SE. Besides that, in view of the kisspeptin is a neuropeptide known for its potent stimulatory action of the gonadotropin-releasing hormone (GNRH), responsible for centrally regulating ovarian function and the estrous cycle. In addition to its fundamental role in the regulation of the reproductive axis, kisspeptin is able to modulate the function of cells in the immune system, with possible regulation in the interaction of these two systems. The present study also aimed to evaluate the modulatory role of kisspeptin in the innate endometrial immune response of bovine females. Therefore, five experiments were carried out and a total of 24 reproductive tracts of nulliparous bovine females were used, collected in a commercial slaughterhouse, for processing through the ex vivo endometrium cultivation technique. The treatments used in the cultivation of endometrial explants from caruncular and intercaruncular regions consisted of kisspeptin (Kp 10-5 M; Kp 10-6 M; Kp 10-7 M), antagonist p234 (p234 10-5 M; p234 10-7 M; p234 10-9 M), progesterone (5ng/mL) and dexamethasone (5ng/mL), challenged or not with 1g/mL lipopolysaccharide (LPS), at 12 and 24 hours of culture. Gene expression of cytokines IL6, IL1 and concentration of cytokines IL6, IL1 and IL8 were evaluated by qRT-PCR and ELISA, respectively. There was no difference in the gene expression of IL1 and IL6 cytokines and in the production of IL6, IL1 and IL8 in any of the treatments performed with different concentrations of kisspeptin. It was concluded that kisspeptin had no modulatory effect on the innate immune response of the bovine endometrium when the ex vivo endometrial culture model was used.

USO DE NANOPARTÍCULAS DE CARBONO EM MODELOS IN VITRO E IN VIVO PARA CONTROLE DA RESPOSTA INFLAMATÓRIA ENDOMETRIAL BOVINA

Nanobiotecnologias baseadas em nanopartículas de carbono para controle de doenças, seja via silenciamento da expressão gênica ou ação anti-inflamatória, surgem como possibilidade para o controle da resposta inflamatória endometrial em fêmeas bovinas no pós-parto. Tendo em vista a possível aplicação de nanotubos de carbono de parede múltipla (MWCNT) em fêmeas bovinas e o desenvolvimento de método de detecção para avaliação da segurança de MWCNT in vivo, a primeira parte do estudo objetivou avaliar a capacidade da técnica de FT-Raman em detectar, qualitativamente e quantitativamente, MWCNT diluídos em leite de vaca in natura. A técnica de FT-Raman descriminou qualitativamente a presença de MWCNT em leite acima de 1g/mL. Entretanto, o modelo de análise de regressão linear não determinou, quantitativamente, diferentes concentrações de MWCNT em leite in natura. Na segunda parte do trabalho, células endometriais bovinas foram submetidas a silenciamento gênico da molécula MyD88, utilizando MWCNT como agente de transcrição, ou cultivadas em meio contendo fulerol ou dexametasona, com o objetivo de controlar a transcrição endometrial de IL-1, IL-6 e CXCL8 induzida in vitro por lipopolissacarídeo (LPS). MWCNT não induziram resposta inflamatória endometrial, porém o complexo silenciador MWCNT-MyD88 siRNA não silenciou a expressão de MyD88 (P>0,05) ou reduziu o número de transcritos para IL-1 (P>0,05), IL-6 (P>0,05) e CXCL8 (P>0,05) pós-estimulação com LPS. Fulerol reduziu o número de transcritos para IL-1 (P<0,01) e dexametasona o número de transcritos para IL-1 (P<0,05) e IL-6 (P<0,05) após estimulação de células endometriais com LPS. O último estudo teve como objetivo verificar, por meio de avaliação histopatológica, a ação anti-inflamatória do fulerol sobre modelo murino in vivo de endometrite induzida por LPS. Entretanto, o modelo proposto não foi efetivo em gerar uma resposta inflamatória endometrial, impossibilitando assim a avaliação da ação anti-inflamatória do fulerol sobre o endométrio murino inflamado. Em conclusão, o uso de MWCNT padronizados como agente de transfecção para siRNA em células endometriais bovinas requer estudos mais aprofundados visando atingir o potencial da técnica no controle da expressão de citocinas pró-inflamatórias. Entretanto, fulerol e dexametasona podem reduzir a expressão de citocinas pró-inflamatórias in vitro, com potencial para controlar, in vivo, a resposta inflamatória endometrial bovina.

Nanobiotechnologies based on carbon nanoparticles for disease control, either by gene silencing or anti-inflammatory action, rise as a possibility for the control of endometrial inflammatory response in post-partum cows. The possibility to apply multi-walled carbon nanotubes (MWCNT) in cow and the development of a methodology to detect carbon nanotubes safety in vivo, the first study aimed to evaluate the qualitative and quantitative potential of the FT-Raman technique to detect MWCNT diluted in bovine whole fresh milk. The FT-Raman qualitatively identified MWCNT in milk over 1g/mL. However, the linear regression analysis model did not quantitatively predict, with precision, different MWCNT dilutions in fresh milk. In the second study, bovine endometrial cells were cultured with MyD88 silencing complex, composed by MWCNT as vector for siRNA targeting MyD88, fullerol, or dexamethasone aiming to control the endometrial transcription of IL-1, IL-6 and CXCL8 induced by lipopolysaccharide (LPS) stimulation in vitro. MWCNT did not induce endometrial inflammatory response. However, the silencing complexes MWCNT-MyD88 siRNA did not silence the MyD88 expression (P>0.05) or reduced the transcript levels for IL-1 (P>0.05), IL-6 (P>0.05) and CXCL8 (P>0.05) post LPS stimulation. Fullerol reduced the transcript level for IL-1 (P<0.01) and dexamethasone decreased transcription of IL-1 (P<0.05) and IL-6 (P<0.05) post endometrial cells stimulation with LPS. The third study aimed to evaluate, by histopathological analysis, the anti-inflammatory effect of fullerol over an in vivo murine model of endometritis induced by LPS. However, the proposed model was not effective to induce an endometrial inflammatory response, avoiding the evaluation of the fullerol anti-inflammatory effect over murine inflamed endometrium. In conclusion, the use standardized MWCNT as transfection agent for siRNA in bovine endometrial cells require more studies aiming to achieve the technique potential for control the expression of pro-inflammatory cytokines. However, fullerol and dexamethasone can reduce the expression of pro-inflammatory cytokines in vitro, with potential to control, in vivo, the endometrial inflammatory response in cattle

Expression of epidermal growth factor (EGF) and its receptor in bovine endometrium throughout the luteal phase: effects of EGF on prostaglandin production in endometrial cells

Epidermal growth factor (EGF) is produced in bovine endometrium throughout the estrous cycle. However, little is known about the expression of the EGF receptor (EGFR) and the roles of EGF in bovine endometrium. To clarify whether EGF is involved in local regulation of bovine endometrial function, first we determined the EGF protein and the expression of EGFR mRNA in endometrial tissues throughout the luteal stages. EGF protein concentration was higher(P < 0.05) in the mid (days 8-12) luteal stage than in the other luteal stages. EGFR mRNA expression was higher (P < 0.05) in the mid and late (days 15-17) luteal stages than in the other luteal stages. To investigate the protein concentrations of EGF and EGFR mRNA expression in cultured bovine endometrial cells, epithelial and stromal cells were isolated between day 0 and day 4 post-ovulation from 22 uteri. Both EGF protein concentration and EGFR mRNA expression were higher (P < 0.05) in epithelial cells than in stromal cells. Then, to examine the possible role of EGF in the regulation of prostaglandin F2α (PGF2α) and prostaglandin E2 (PGE2), cultured endometrial epithelial and stromal cells were exposed to EGF (0, 1, 10 and 100 nm) for 24 h. In epithelial cells, EGF (10 and/or 100 nm) increased (P < 0.05) PGF2α and PGE2 secretion, but in stromal cells EGF (100 nm) increased (P < 0.05) PGF2α, but not PGE2 secretion. These results indicate that 1) the highest amount of EGF is produced by bovine endometrium at the mid-luteal stage, 2) endometrial EGFR mRNA expressions are higher at mid and late-luteal stages than other stages, 3) EGF is expressed mainly by uterine epithelial cells and 4) EGF has the ability to increase PGE2 and PGF2α production in both epithelial and stromal cells and therefore may play a role in local regulation of uterine function.

Expression of epidermal growth factor (EGF) and its receptor in bovine endometrium throughout the luteal phase: effects of EGF on prostaglandin production in endometrial cells

Epidermal growth factor (EGF) is produced in bovine endometrium throughout the estrous cycle. However, little is known about the expression of the EGF receptor (EGFR) and the roles of EGF in bovine endometrium. To clarify whether EGF is involved in local regulation of bovine endometrial function, first we determined the EGF protein and the expression of EGFR mRNA in endometrial tissues throughout the luteal stages. EGF protein concentration was higher(P < 0.05) in the mid (days 8-12) luteal stage than in the other luteal stages. EGFR mRNA expression was higher (P < 0.05) in the mid and late (days 15-17) luteal stages than in the other luteal stages. To investigate the protein concentrations of EGF and EGFR mRNA expression in cultured bovine endometrial cells, epithelial and stromal cells were isolated between day 0 and day 4 post-ovulation from 22 uteri. Both EGF protein concentration and EGFR mRNA expression were higher (P < 0.05) in epithelial cells than in stromal cells. Then, to examine the possible role of EGF in the regulation of prostaglandin F2α (PGF2α) and prostaglandin E2 (PGE2), cultured endometrial epithelial and stromal cells were exposed to EGF (0, 1, 10 and 100 nm) for 24 h. In epithelial cells, EGF (10 and/or 100 nm) increased (P < 0.05) PGF2α and PGE2 secretion, but in stromal cells EGF (100 nm) increased (P < 0.05) PGF2α, but not PGE2 secretion. These results indicate that 1) the highest amount of EGF is produced by bovine endometrium at the mid-luteal stage, 2) endometrial EGFR mRNA expressions are higher at mid and late-luteal stages than other stages, 3) EGF is expressed mainly by uterine epithelial cells and 4) EGF has the ability to increase PGE2 and PGF2α production in both epithelial and stromal cells and therefore may play a role in local regulation of uterine function.(AU)

ÁCIDOS GRAXOS NÃO ESTERIFICADOS (NEFAs) E BETA-HIDROXIBUTIRATO (BHBA) NA REGULAÇÃO DA FOLICULOGÊNESE E EPIGENÉTICA DAS CÉLULAS ENDOMETRIAIS

Um dos principais problemas abordados na pecuária de leite nas últimas décadas tem sido a redução da fertilidade em vacas de alta produção. O balanço energético negativo (BEN) durante o período pós-parto é um fator de risco para que ocorram alterações reprodutivas. Durante esse período, as concentrações séricas dos ácidos graxos não esterificados (NEFAs) e do beta-hidroxibutirato (BHBA) estão aumentadas. Essas alterações têm reflexo no microambiente folicular e endométrio bovino. Além disso, o exato mecanismo de ação dos NEFAs e BHBA nos tecidos-alvo ainda não está estabelecido, podendo ocorrer via receptores nucleares. Portanto, o objetivo do presente trabalho foi investigar a ação dos NEFAs e do BHBA na foliculogênese e no endométrio bovino, bem como investigar se os efeitos ocorrem via receptor ativado por proliferador de peroxissomo gama (PPARG). Primeiramente, investigou-se a participação do PPARG na regulação do desenvolvimento do folículo dominante e ovulação. Os resultados deste estudo revelaram que a abundância de RNAm do PPARG foi similar entre os folículos dominante e subordinado em torno da divergência folicular, diminuindo após o pico de LH e aumentando antes da ovulação. Além disso, a ativação do PPARG com Troglitazone (TGZ; agonista do PPARG) inibiu o crescimento folicular e diminuiu a expressão de RNAm da CYP19A1 nas células da granulosa bovina. Portanto, PPARG é envolvido na regulação da esteroidogênese, crescimento folicular e ovulação em bovinos. O segundo estudo teve como objetivo investigar se a injeção intrafolicular com os três principais NEFAs (ácido oleico, esteárico e palmítico) afeta o desenvolvimento do folículo dominante e a expressão de RNAm de genes candidatos nas células da granulosa. Observou-se que a injeção intrafolicular com os NEFAs reduziram o crescimento folicular 24 e 48 h após o tratamento, demonstrando que altas concentrações dos NEFAs são prejudiciais para o desenvolvimento folicular. No entanto, a expressão de genes relacionados a esteroidogênese, metabolismo e apoptose não foram afetados. Da mesma forma, a expressão de RNAm do PPARG não foi alterada após o tratamento. Portanto, os NEFAs afetam a taxa de crescimento folicular do folículo dominante, mas o mecanismo de ação ainda necessita mais estudos. O terceiro estudo teve como objetivo avaliar os efeitos dos NEFAs e BHBA nas células endometriais bovinas. Os resultados obtidos revelaram que os tratamentos in vitro com NEFAs e NEFAs+BHBA induziram um acúmulo de lipídeos nas células, estimularam a produção de espécies reativas ao oxigênio (ROS), alteraram a morfologia das células e aumentaram a expressão de RNAm dos genes relacionados ao estresse oxidativo, apoptose e transporte de glicose. O estudo também evidenciou que o ácido palmítico (AP) afeta a regulação epigenética nas células endometriais, alterando a trimetilação da lisina 4 da histona 3 (H3K4me3) e a expressão de RNAm da DNA metiltransferase 1 (DNMT1). Em conjunto, os dados apresentados evidenciam os efeitos deletérios do aumento dos NEFAs e BHBA no desenvolvimento folicular e nas células endometriais bovinas.

Dairy cows milk production has increased over the past decades as a result of intense genetic selection. However, the advances in genetic selection of dairy herds did not come with an improvement of reproductive parameters. Negative energy balance (NEB) during postpartum period is important risk factor in the establishment of reproductive failure in high producing dairy cows. During NEB, nonesterified fatty acids (NEFAs) and -hydroxybutyrate (BHBA) levels are increased in the serum and follicular fluid and affect follicular microenvironment and endometrium in cattle. Furthermore, the exact NEFAs and BHBA mechanism of action is not yet established and may occur via nuclear receptors. Therefore, the aim of present study was to investigate NEFAs and BHBA action on folliculogenesis and endometrium, as well as to investigate if peroxisome proliferator activated receptor gamma (PPARG) is involved in their regulation in cattle. In the first study, the aim was to investigate if the PPARG participates in the regulation of dominant follicle development and ovulation. Findings from this study revealed that the relative mRNA abundance of PPARG was similar between dominant and subordinate follicles around follicle deviation, decreased after the LH surge, and increased before ovulation. Intrafollicular injection of Troglitazone (TGZ; a PPARG agonist) inhibited follicular growth and decreased CYP19A1 mRNA abundance in granulosa cells. These findings indicate that PPARG is involved in the regulation of steroidogenesis, follicle growth and ovulation in cattle. In a second study, the aim was to investigate if intrafolicular injection of three most important NEFAs (oleic, stearic and palmitic acids) affects dominant follicle development and mRNA expression of candidate genes in granulosa cells. NEFAs injection reduced follicular growth at 24 and 48 h, demonstrating that the high concentrations of NEFAs are detrimental to follicle development. The mRNA expression of genes related to steroidogenesis energetic metabolism and apoptosis examined were not affected by intrafollicular injection of NEFAs. However, NEFAs affected the follicle growth rate of dominant follicle but the mechanism of action still needs further study. In the final study, we evaluated the effects of NEFAs and BHBA in endometrium cells. Findings from this study showed that in vitro treatment with NEFAs and NEFAs plus BHBA increased lipid accumulation, stimulated reactive oxygen species (ROS), altered the morphology and the mRNA expression of genes related to oxidative stress, apoptosis and glucose transport in the endometrium cells. Also, palmitic acid (PA) was able to modulate the try-methylation of histone 3 lysine 4 (H3K4me3) and the mRNA expression of DNA methyltransferase 1 (DNMT1). Altogether, data present in these studies provided evidence that NEFAs and BHBA are detrimental to follicle development and endometrium cells in cattle.

Produção de IL-6 e IL-1 em cultivos ex vivo de endométrio bovino inoculados com Leptospira interrogans sorovar Hardjoprajitno

Leptospirose é uma zoonose causada por bactérias do gênero Leptospira spp. Existem relatos de isolamento desta bactéria no trato genital de fêmeas, relacionado à abortamento e infertilidade no rebanho bovino. Já se sabe que a Leptospira presente nos órgãos reprodutivos de bovinos causa danos teciduais principalmente pela vasculite. Além disso, a leptospirose aguda produz níveis altos de citocinas durante a fase inicial de infecção. Entretanto, faltam estudos sobre a patogênese desse microrganismo no endométrio de vacas. Objetivou-se avaliar o perfil das citocinas inflamatórias após o desafio com Leptospira interrogans sorovar Hardjoprajitno e ao LPS de Escherichia coli produzidas pelo tecido uterino ex vivo coletados de vacas não gestantes e gestantes reativas sorologicamente ou não ao Teste de soroaglutinação microscópica (MAT). Foram utilizados 43 úteros com respectivas amostras de sangue coletadas de vacas mestiças, sendo 19 delas não gestantes (19/43) e 24 gestantes (24/43), todas sem evidência de doença clínica. Para determinação da fase gestacional foi realizada através das medidas dos fetos. Para análise de uma prévia infecção foram avaliados anticorpos antiLeptospira,por meio do teste de aglutinação microscópica (MAT). Após a exposição do endométrio,com auxílio de um punch de 8 mm foram retirados fragmentos de endométrio (explantes)foram retirados nas regiões intercarunculares. O processamento dos explantes endometriais ocorreu em placas de cultura celular de 24 poços. As soluções adicionadas sobre os explantes foram as seguintes: a) meio controle, contendo 2 mL de solução de RPMI; b) meio RPMI, contendo 1g/mL de LPS de Escherichia coli; c) meio EMJH com inóculo de Leptospira interrogans sorovar Hardjoprajitno na concentração de 108 leptospiras por mL. Em seguida,as placas foram incubadas em estufa a 37ºC com 5% de CO2 permanecendo por 24 horas. Após esse período, os tecidos endometriais foram pesados e os sobrenadantes foram recolhidos e subsequentemente testados para determinação dos perfis de produção das interleucinas 1 e 6 pelo Kits de ELISA. Em relação ao desafio com 1g/mL de LPS, tanto explantes das vacas não gestantes como gestantes montaram uma resposta frente ao desafio, com aumento significativo da produção de IL-6 e IL-1 em relação aos controles. No desafio com Leptospira, os explantes de vacas não gestantes e gestantes, houve aumento na concentração de IL-1. Entretanto, somente explantes das vacas não gestantes houve aumento de IL-6 em relação aos controles. Na avaliação individual das fases de gestação, somente na fase inicial (primeiro trimestre) ocorreu produção de IL-6 significativa, diferente do observado com IL-1, sendo que ocorreu diferença significativa na produção desta citocina na fase final de gestação, no desafio com 1g/mL de LPS de E.coli. Já no desafio com Leptospira não houve diferença significativa entre as fases gestacionais. Conclui-se que ocorre maior estimulação da produção de citocinas do endometrio de vacas reagentes, nos desafios ao LPS e à Leptospira; menor estimulação da produção de IL-6 ao desafio com Leptospira em vacas gestantes; e maior estimulação na fase inicial e final de gestação no desafio com LPS de E.coli e nenhuma diferença significativa entre as fases gestacionais no desafio com Leptospira.

Leptospirosis is a zoonosis caused by a bacteria belonging to genus Leptospira spp. There are reports of this bacteria isolation in the female reproductive tract with are related to abortion and subfertility in bovine herds. It is known that Leptospira presence in reproductive organs of bovines promotes tissue damages especially by vasculitis. Moreover, acute leptospirosis produces high levels of cytokines during the onset of infection. However, few studies were performed about the pathogenesis of this microorganism in the endometrium of cows. The objective of this study was to evaluate the inflammatory cytokines profile after Leptospira interrogans serovars Hardjoprajitno and LPS of Escherichia coli challenges produced by uterine tissue ex vivo of non-pregnant and pregnant cows serologically reactive or not to Microscopic Agglutination Test (MAT). A total of 43 uterus with respective blood samples were collected from crossbred cows, from those 19 were non-pregnant and 24 pregnant, without any clinical disease evidence. To determine the pregnancy phase, the measurement of fetuses were performed. To analyze previous infection occurrence antibodies against Leptospira were evaluated by MAT. After endometrium exposure, fragments of endometrium tissue (explants) were collected using a punch of 8 mm in intercaruncular region. The processing of endometrial explants were done in a 24-well cell culture plates. The solutions added on explants were: a) Control medium (2 mL of RPMI solution); b) RPMI medium (1g/mL of LPS from Escherichia coli); c) EMJH medium with inoculum of Leptospira interrogans serovars Hardjoprajitno (108 leptospires/mL). The plates were incubated at 37°C with 5% of CO2 for 24 hours. After this period, uterine explants were weighed and collected and subsequently tested for interleukins (IL-1 and IL-6) production profile by ELISA kits. Considering the challenge with 1g/mL of LPS, both pregnant and non-pregnant cows build a response due to challenge with a significant increase in IL-1 and IL-6 production compared to control group. Considering the challenge with Leptospira, an increase of IL-6 concentration was verified in the explants of both pregnant and non-pregnant cows related to control. However, only the explants from non-pregnant cows showed an increase of IL-6 production related to control. In the individual evaluation of pregnancy phases, during the initial phase (first trimester) only occurred significant IL-6 production, differing from IL-1 which significant production increase occurred during late gestation phase in the challenge with 1g/mL of LPS from E.coli. There was no difference in cytokines production according to pregnancy phase after challenge with Leptospira. In conclusion, a higher stimulation of cytokines production was verified in endometrial tissues of reagent cows after LPS and Leptospira challenges; lower stimulation of IL-6 production in pregnant cows after Leptospira challenge; a higher stimulation during initial and late pregnancy phases after LPS from E.coli challenge, and no significant difference between pregnancy phases after Leptospira challenge.