Cell differentiation events in pre-implantation mouse and bovine embryos

Abstract Early mammal embryogenesis starts with oocyte fertilization, giving rise to the zygote. The events that the newly formed zygote surpasses are crucial to the embryo developmental success. Shortly after activation of its genome, cells of the embryo segregate into the inner cell mass (ICM) or the trophectoderm (TE). The first will give rise to the embryo while the latter will become the placenta. This first segregation involves cellular and molecular processes that include cell polarity linked to intracellular pathway activation, which will regulate the transcription of trophectoderm-related genes. Then, cells of the ICM undergo the second event of mammalian cell differentiation, which consists of the separation between epiblast (EPI) and hypoblast or primitive endoderm (PrE). This second segregation involves paracrine signaling, leading to differential expression of key genes that will dictate the fate of the cell. Although these processes are described in detail in the mouse, recent studies suggest that the bovine embryo could also be an interesting model for early development, since there are differences to the mouse and similarities with early human embryogenesis. In this review, we gathered the main data available in the literature upon bovine and mouse early development events, suggesting that both models should be analyzed and studied in a complementary way, to better model early events occurring in human development.

Cell differentiation events in pre-implantation mouse and bovine embryos

Early mammal embryogenesis starts with oocyte fertilization, giving rise to the zygote. The events that the newly formed zygote surpasses are crucial to the embryo developmental success. Shortly after activation of its genome, cells of the embryo segregate into the inner cell mass (ICM) or the trophectoderm (TE). The first will give rise to the embryo while the latter will become the placenta. This first segregation involves cellular and molecular processes that include cell polarity linked to intracellular pathway activation, which will regulate the transcription of trophectoderm-related genes. Then, cells of the ICM undergo the second event of mammalian cell differentiation, which consists of the separation between epiblast (EPI) and hypoblast or primitive endoderm (PrE). This second segregation involves paracrine signaling, leading to differential expression of key genes that will dictate the fate of the cell. Although these processes are described in detail in the mouse, recent studies suggest that the bovine embryo could also be an interesting model for early development, since there are differences to the mouse and similarities with early human embryogenesis. In this review, we gathered the main data available in the literature upon bovine and mouse early development events, suggesting that both models should be analyzed and studied in a complementary way, to better model early events occurring in human development.(AU)

Morphometry of bovine blastocysts produced in vitro in culture media with antioxidants cysteamine or oily extract of Lippia origanoides / [Morfometria de blastocistos bovinos produzidos in vitro em meio de cultura com antioxidantes cisteamina ou extrato oleoso de Lippia origanoides]

This study aimed to evaluate the ultrastructural morphometry of bovine embryos produced in vitro grown at different concentrations of antioxidants. After in vitro maturation and fertilization, the presumptive zygotes were assigned into five treatments. T1) without the addition of any antioxidants (negative control); T2) addition of 50µM/mL cysteamine; and T3, T4 and T5) adding 2.5µg/mL, 5.0µg/mL or 10.0µg/mL of the antioxidants derived from the oily extract from Lippia origanoides, respectively. On D7 of culture, the embryos in the blastocyst stage were fixed and prepared for electron transmission microscopy. These were evaluated for the proportion of cytoplasm-to-nucleus, cytoplasm-to-mitochondria, cytoplasm-to-vacuoles, cytoplasm-to-autophagic vacuoles and cytoplasm-to-lipid droplets. Blastocysts cultured in media containing oily extract of Lippia origanoides presented morphological characteristics such as high cell:mitochondria ratio and low cell:vacuoles and cell:autophagic vacuole ratio, possibly been morphological indicators of embryonic quality. Inner cell mass (ICM) from blastocysts cultured in media without any antioxidants had the highest cell:vacuole ratio. Similar results were found in the trophectoderm (TE) cells of blastocysts from treatment 2. Embryo culture media supplemented with antioxidants derived from Lippia origanoides oil produced embryos with a higher cytoplasmic proportion of organelles, such as mitochondria. Also, treatments without any antioxidants or with the addition of cysteamine presented cytoplasmic vacuolization, a characteristic related to production of poor-quality embryos.(AU)

Este estudo teve como objetivo avaliar a morfometria ultraestrutural de embriões bovinos produzidos in vitro e cultivados em diferentes concentrações de antioxidantes. Após a maturação e a fertilização in vitro, os possíveis zigotos foram divididos em cinco tratamentos: T1) sem adição de antioxidantes (controle negativo); T2) adição de 50µM/mL de cisteamina; e T3, T4 e T5) adição de 2,5µg/mL, 5,0µg/mL ou 10,0µg/mL dos antioxidantes derivados do extrato oleoso de Lippia origanoides, respectivamente. No D7 de cultivo, os embriões em estágio de blastocisto foram fixados e preparados para microscopia eletrônica de transmissão. Estes foram avaliados para a proporção entre citoplasma e núcleo, citoplasma e mitocôndria, citoplasma e vacúolos, citoplasma e vacúolos autofágicos e citoplasma e gotículas lipídicas. Blastocistos cultivados em meio contendo extrato oleoso de Lippia origanoides apresentaram características morfológicas como alta relação célula:mitocôndria e baixa relação célula:vacúolos e célula:vacúolo autofágico, possíveis indicadores morfológicos de qualidade embrionária. A massa celular interna (MCI) de blastocistos cultivados em meio sem quaisquer antioxidantes teve a maior razão célula:vacúolo. Resultados semelhantes foram encontrados nas células do trofectoderma (TE) de blastocistos do tratamento 2. Portanto, o meio de cultivo embrionário suplementado com antioxidantes derivados do óleo de Lippia origanoides produziu embriões com maior proporção citoplasmática de organelas, como mitocôndrias. Além disso, tratamentos sem antioxidantes ou com adição de cisteamina apresentaram vacuolização citoplasmática, característica relacionada à produção de embriões de baixa qualidade.(AU)

Cellular events during ovine implantation and impact for gestation

The establishment of pregnancy in sheep includes elongation of the blastocyst into a filamentous conceptus, pregnancy recognition, production of histotroph, attachment of the conceptus to the endometrium for implantation, and development of synepitheliochorial placentation. These processes are complex, and this review describes some of the molecular events that underlie and support successful pregnancy. The free-floating sheep blastocyst elongates into a filamentous conceptus and metabolizes, or is responsive to, molecules supplied by the endometrium as histotroph. Amongst these molecules are SPP1, glucose and fructose, and arginine that stimulate the MTOR nutrient sensing system. The placental trophectoderm of elongating conceptuses initiate pregnancy recognition and implantation. The mononucleate cells of the trophectoderm secrete IFNT, which acts on the endometrial LE to block increases in estrogen receptor α to preclude oxytocin receptor expression, thereby preventing oxytocin from inducing luteolytic pulses of PGF2α. In addition, IFNT increases expression of IFN stimulated genes in the endometrial stroma, including ISG15, a functional ubiquitin homologue. Implantation is the initial step in placentation, and includes sequential pre-contact, apposition, and adhesion phases. Implantation in sheep includes downregulation of Muc1 and interaction of GLYCAM1, galectin 15 (LGALS15) and SPP1 with lectins and integrins (αvβ3). Sheep have synepitheliochorial placentation in which mononucleate trophectoderm cells fuse to form binucleate cells (BNCs). BNCs migrate and fuse with endometrial LE cells to form trinucleate syncytial cells, and these syncytia enlarge through continued BNC fusion to form syncytial plaques that form the interface between endometrial and placental tissues within the placentome. The placentae of sheep organize into placentomal and interplacentomal regions. In placentomes there is extensive interdigitation of endometrial and placental tissues to provide hemotrophic nutrition to the fetus. In interplacentomal regions there is epitheliochorial attachment of endometrial LE to trophectoderm, mediated through focal adhesion assembly, and areolae that take up histotroph secreted by endometrial GE.

Cellular events during ovine implantation and impact for gestation

The establishment of pregnancy in sheep includes elongation of the blastocyst into a filamentous conceptus, pregnancy recognition, production of histotroph, attachment of the conceptus to the endometrium for implantation, and development of synepitheliochorial placentation. These processes are complex, and this review describes some of the molecular events that underlie and support successful pregnancy. The free-floating sheep blastocyst elongates into a filamentous conceptus and metabolizes, or is responsive to, molecules supplied by the endometrium as histotroph. Amongst these molecules are SPP1, glucose and fructose, and arginine that stimulate the MTOR nutrient sensing system. The placental trophectoderm of elongating conceptuses initiate pregnancy recognition and implantation. The mononucleate cells of the trophectoderm secrete IFNT, which acts on the endometrial LE to block increases in estrogen receptor α to preclude oxytocin receptor expression, thereby preventing oxytocin from inducing luteolytic pulses of PGF2α. In addition, IFNT increases expression of IFN stimulated genes in the endometrial stroma, including ISG15, a functional ubiquitin homologue. Implantation is the initial step in placentation, and includes sequential pre-contact, apposition, and adhesion phases. Implantation in sheep includes downregulation of Muc1 and interaction of GLYCAM1, galectin 15 (LGALS15) and SPP1 with lectins and integrins (αvβ3). Sheep have synepitheliochorial placentation in which mononucleate trophectoderm cells fuse to form binucleate cells (BNCs). BNCs migrate and fuse with endometrial LE cells to form trinucleate syncytial cells, and these syncytia enlarge through continued BNC fusion to form syncytial plaques that form the interface between endometrial and placental tissues within the placentome. The placentae of sheep organize into placentomal and interplacentomal regions. In placentomes there is extensive interdigitation of endometrial and placental tissues to provide hemotrophic nutrition to the fetus. In interplacentomal regions there is epitheliochorial attachment of endometrial LE to trophectoderm, mediated through focal adhesion assembly, and areolae that take up histotroph secreted by endometrial GE.(AU)

Physiological and cellular requirements for successful elongation of the preimplantationconceptus and the implications for fertility in lactating dairy cows

Elongation of the preimplantation conceptus is a prerequisite for maternal recognition of pregnancy and implantation in ruminants. Failures in this phase of development likely contribute for the subfertility of lactating dairy cows. This review will discuss our current understanding of the physiological and cellular requirements for successful elongation of the preimplantation conceptus and their potential deficiency in subfertile lactating dairy cows. Major requirements include the priming of the endometrium by ovarian steroids, reprogramming of trophectoderm cells at the onset of elongation, and intensification of the crosstalk between elongating conceptus and endometrium. Conceptus elongation and survival in dairy cows does not seem to be affected by lactation per se but seem to be altered in subgroups of cows with endocrine, metabolic and nutritional imbalances or deficiencies. These subgroups of cows include those suffering diseases postpartum, anovular cows enrolled in synchronization programs, and cows with low concentration of circulating steroids and IGF1. Success of conceptus elongation starts long before breeding and entails optimization of health and nutrition programs, especially during the transition period, and might be extended to the supplementation of endocrine and nutritional shortages at the time of breeding. Genetic selection will eventually become more important as researchers unravel the molecular control of reproduction and develop new fertility traits focused on pregnancy survival.

Physiological and cellular requirements for successful elongation of the preimplantationconceptus and the implications for fertility in lactating dairy cows

Elongation of the preimplantation conceptus is a prerequisite for maternal recognition of pregnancy and implantation in ruminants. Failures in this phase of development likely contribute for the subfertility of lactating dairy cows. This review will discuss our current understanding of the physiological and cellular requirements for successful elongation of the preimplantation conceptus and their potential deficiency in subfertile lactating dairy cows. Major requirements include the priming of the endometrium by ovarian steroids, reprogramming of trophectoderm cells at the onset of elongation, and intensification of the crosstalk between elongating conceptus and endometrium. Conceptus elongation and survival in dairy cows does not seem to be affected by lactation per se but seem to be altered in subgroups of cows with endocrine, metabolic and nutritional imbalances or deficiencies. These subgroups of cows include those suffering diseases postpartum, anovular cows enrolled in synchronization programs, and cows with low concentration of circulating steroids and IGF1. Success of conceptus elongation starts long before breeding and entails optimization of health and nutrition programs, especially during the transition period, and might be extended to the supplementation of endocrine and nutritional shortages at the time of breeding. Genetic selection will eventually become more important as researchers unravel the molecular control of reproduction and develop new fertility traits focused on pregnancy survival.(AU)

Effect of follicular diameter, time of first cleavage and H3K4 methylation on embryo production rates of Bos indicus cattle / Efeito do diâmetro folicular, momento da primeira clivagem e metilação da H3K4 na produção embrionária de vacas Bos indicus

This study aimed investigate the relationship between epigenetics, follicular diameter and cleavage speed, by evaluating the developmental potential and occurence of H3K4 monomethylation of early-, intermediate- and late-cleaving Bos indicus embryos from in vitro fertilized oocytes originating from follicles up to 2 mm in diameter or between 4 and 8 mm in diameter. Oocytes (n = 699) from small follicles (? 2 mm) and 639 oocytes from large follicles (4-8 mm) were punched from 1,982 Bos indicus slaughterhouse ovaries. After maturation and in vitro fertilization (IVF), the cultured embryos were separated into early (? 28 h post-IVF), intermediate (> 28 h and ? 34 h post-IVF) and late (> 34 h and ? 54 h post-IVF) cleavage groups. Blastocysts were subjected to an immunofluorescence assessment for H3K4me investigation. The blastocyst rate for large follicles (36.3%) was higher than that for small follicles (22.9%, P 0.05). In addition, blastocyst rates for early and intermediate cleavage groups (45.3% and 33.8%, respectively) were higher than that for late cleavage group (13.5%, P 0.05). The blastocysts from all groups displayed H3K4me staining by immunofluorescence, particularly intense in what seemed to be trophectoderm cells and weak or absent in cells seemingly from the inner cell mass. For the first time for indicus embryos, data from this study demonstrate that higher blastocyst embryorates are obtained from embryos that cleave within 34 h after fertilization and from those produced fromfollicles of 4-8 mm in diameter, indicating a greater ability of these embryos to develop to the stage ofembryonic preimplantation. This is the first article demonstrating the occurrence of H3K4me in cattleembryos; its presence in all the evaluated blastocysts suggests that this histone modification plays a keyrole in maintaining embryo viability at preimplantation stage.

Este estudo teve como objetivo investigar a relação entre a epigenética, o diâmetro folicular e a velocidade de clivagem, avaliando o potencial de desenvolvimento e a ocorrência de monometilação da H3K4 em embriões Bos indicus de clivagem precoce, intermediária e tardia produzidos a partir de oócitos fertilizados in vitro oriundos de folículos de até 2 mm de diâmetro ou entre 4 e 8 mm de diâmetro. Oócitos (n = 699) de folículos pequenos (? 2 mm) e 639 oócitos de folículos grandes (4-8 mm) foram puncionados de 1982 ovários de vacas Bos indicus de abatedouro. Após a maturação e fertilização in vitro (FIV), os embriões cultivados foram separados nos grupos de clivagem precoce (? 28 h pós-FIV), intermediária (> 28 h e ? 34 h pós-FIV) e tardia (> 34 h e ? 54 h pós-FIV). Os blastocistos foram submetidos à imunofluorescência para investigação de H3K4me. A taxa de blastocisto para embriões provenientes de folículos grandes (36,3%) foi maior que de folículos pequenos (22,9%; p 0,05). Ainda, as taxas de blastocisto para os grupos de clivagem precoce e intermediária (45,3% e 33,8%, respectivamente) foram maiores que para o grupo de clivagem tardia (13,5%; p 0,05). Blastocistos de todos os grupos mostraram marcação para H3K4me à imunofluorescência, particularmente intensa no que pareciam ser células do trofectoderma e fraca ou ausente em células semelhantes às da massa massa celular interna. Pela primeira vez em embriões indicus, os dados deste estudo demonstram que maiores taxas deblastocisto são obtidas de embriões que clivam em até 34 h pós-fertilização e dos oriundos de folículos de 4 a 8 mm de diâmetro, indicando uma maior habilidade desses embriões de se desenvolverem até o estágio de pré-implantação embrionária.

Effect of follicular diameter, time of first cleavage and H3K4 methylation on embryo production rates of Bos indicus cattle / Efeito do diâmetro folicular, momento da primeira clivagem e metilação da H3K4 na produção embrionária de vacas Bos indicus

This study aimed investigate the relationship between epigenetics, follicular diameter and cleavage speed, by evaluating the developmental potential and occurence of H3K4 monomethylation of early-, intermediate- and late-cleaving Bos indicus embryos from in vitro fertilized oocytes originating from follicles up to 2 mm in diameter or between 4 and 8 mm in diameter. Oocytes (n = 699) from small follicles (? 2 mm) and 639 oocytes from large follicles (4-8 mm) were punched from 1,982 Bos indicus slaughterhouse ovaries. After maturation and in vitro fertilization (IVF), the cultured embryos were separated into early (? 28 h post-IVF), intermediate (> 28 h and ? 34 h post-IVF) and late (> 34 h and ? 54 h post-IVF) cleavage groups. Blastocysts were subjected to an immunofluorescence assessment for H3K4me investigation. The blastocyst rate for large follicles (36.3%) was higher than that for small follicles (22.9%, P 0.05). In addition, blastocyst rates for early and intermediate cleavage groups (45.3% and 33.8%, respectively) were higher than that for late cleavage group (13.5%, P 0.05). The blastocysts from all groups displayed H3K4me staining by immunofluorescence, particularly intense in what seemed to be trophectoderm cells and weak or absent in cells seemingly from the inner cell mass. For the first time for indicus embryos, data from this study demonstrate that higher blastocyst embryorates are obtained from embryos that cleave within 34 h after fertilization and from those produced fromfollicles of 4-8 mm in diameter, indicating a greater ability of these embryos to develop to the stage ofembryonic preimplantation. This is the first article demonstrating the occurrence of H3K4me in cattleembryos; its presence in all the evaluated blastocysts suggests that this histone modification plays a keyrole in maintaining embryo viability at preimplantation stage.(AU)

Este estudo teve como objetivo investigar a relação entre a epigenética, o diâmetro folicular e a velocidade de clivagem, avaliando o potencial de desenvolvimento e a ocorrência de monometilação da H3K4 em embriões Bos indicus de clivagem precoce, intermediária e tardia produzidos a partir de oócitos fertilizados in vitro oriundos de folículos de até 2 mm de diâmetro ou entre 4 e 8 mm de diâmetro. Oócitos (n = 699) de folículos pequenos (? 2 mm) e 639 oócitos de folículos grandes (4-8 mm) foram puncionados de 1982 ovários de vacas Bos indicus de abatedouro. Após a maturação e fertilização in vitro (FIV), os embriões cultivados foram separados nos grupos de clivagem precoce (? 28 h pós-FIV), intermediária (> 28 h e ? 34 h pós-FIV) e tardia (> 34 h e ? 54 h pós-FIV). Os blastocistos foram submetidos à imunofluorescência para investigação de H3K4me. A taxa de blastocisto para embriões provenientes de folículos grandes (36,3%) foi maior que de folículos pequenos (22,9%; p 0,05). Ainda, as taxas de blastocisto para os grupos de clivagem precoce e intermediária (45,3% e 33,8%, respectivamente) foram maiores que para o grupo de clivagem tardia (13,5%; p 0,05). Blastocistos de todos os grupos mostraram marcação para H3K4me à imunofluorescência, particularmente intensa no que pareciam ser células do trofectoderma e fraca ou ausente em células semelhantes às da massa massa celular interna. Pela primeira vez em embriões indicus, os dados deste estudo demonstram que maiores taxas deblastocisto são obtidas de embriões que clivam em até 34 h pós-fertilização e dos oriundos de folículos de 4 a 8 mm de diâmetro, indicando uma maior habilidade desses embriões de se desenvolverem até o estágio de pré-implantação embrionária. (AU)

Inserção de gene repórter fluorescente em região gênica de SOX2 para estudo da diferenciação celular no desenvolvimento embrionário inicial bovino

A separação entre massa interna celular (MCI) e trofectoderma (TE) é o primeiro evento de diferenciação celular que ocorre no embrião mamífero, seguida pela diferenciação na MCI do epiblasto (EPI) e endoderma primitivo (PE). Falhas nestes processos de desenvolvimento inicial podem ocasionar perdas embrionárias e consequentemente econômicas. A influência do sistema de produção embrionária nos primeiros eventos de diferenciação celular precisa ser mais elucidada, refletindo em índices que ainda podem ser melhorados na produção de embriões bovinos in vitro. O soro fetal bovino (SFB), apesar de seus efeitos deletérios sobre fatores epigenéticos, ainda é usado em sistemas de produção in vitro e pode influenciar esta diferenciação. Ainda, as técnicas para estudo dos embriões normalmente envolvem a destruição dos mesmos e não permitem acompanhar em tempo real a dinâmica de expressão dos genes relacionados à diferenciação em MCI e TE. Novas técnicas como CRISPR/Cas9 permitem alteração direcionada da sequência de DNA genômico, assim, a introdução direcionada de proteínas fluorescentes repórteres permitiria a visualização em tempo real da expressão de genes de interesse nos embriões. Diante disso, nossos objetivos foram: testar a influência do soro fetal bovino nas primeiras diferenciações e, realizar a inserção dirigida de uma proteína repórter fluorescente na região do gene SOX2 por recombinação homóloga. Ainda, comparamos a eficiência da inserção gênica com uso do sistema CRISPR em diferentes momentos do desenvolvimento embrionário, com diferentes horários pós inseminação e com diferentes concentrações de material injetado. No primeiro experimento observamos que a retirada do SFB do cultivo embrionário não causa alterações na alocação de células na MCI ou TE, quando se alia esta remoção à renovação de uma parte do meio de cultivo às 90 horas pós inseminação (hpi). A ausência de suplementação de SFB ou de meio de cultivo levou à diminuição das células do TE, entretanto não alterou o número de células do PE. No segundo experimento, nenhum embrião produzido apresentou fluorescência, portanto todos os blastocistos tiveram seu DNA extraído para genotipagem. Foi realizada uma amostragem, e de 34 embriões verificados, um apresentou banda correspondente ao local do inserto e também banda correspondente ao embrião controle (wild type), sugerindo mosaicismo. Ao final, observamos que as melhores probabilidades de inserção gênica nos embriões bovinos aconteceram quando a injeção dos componentes do sistema CRISPR foi realizada após 8 horas de FIV e com maiores concentrações. Em conclusão, pudemos remover o SFB do nosso sistema de produção de embriões e surpreendentemente o SFB não influenciou a diferenciação em PE. Ainda, são necessários maiores estudos a fim de se otimizar a ferramenta para a obtenção de knock-in em embriões bovinos, principalmente para que se obtenha taxas de maior sucesso e redução da probabilidade de ocorrência de mosaicismo nestes embriões.

The separation between inner cell mass (ICM) and trophectoderm (TE) is the first cell differentiation event that occurs in the mammalian embryo, followed by differentiation into the epiblast MCI (EPI) and primitive endoderm (PE). Failures in these early development processes can lead to embryonic and subsequent economic losses. The influence of the embryo production system on the first cell differentiation events needs to be further elucidated, reflecting in rates that can still be improved in the production of in vitro bovine embryos. Fetal bovine serum (FBS), despite its deleterious effects on epigenetic factors, is still used in in vitro production systems and may influence this differentiation. Furthermore, the techniques for studying embryos usually involve their destruction and do not allow real-time monitoring of the expression dynamics of genes related to differentiation in ICM and TE. New techniques such as CRISPR/Cas9 allow targeted alteration of genomic DNA sequence, thus targeted introduction of fluorescent reporter proteins would allow real-time visualization of the expression of genes of interest in embryos. Therefore, our objectives were: to test the influence of fetal bovine serum in the first differentiations and to carry out the targeted insertion of a fluorescent reporter protein in the region of the SOX2 gene by homologous recombination. Furthermore, we compared the efficiency of gene insertion using the CRISPR system at different moments of embryonic development, with different times after insemination and with different concentrations of injected material. In the first experiment, we observed that FBS removal from the embryonic culture does not cause changes in cell allocation in the ICM or TE, when this removal is combined with the renewal of a part of the culture medium at 90 hours after insemination (hpi). The absence of FBS or culture medium supplementation led to a decrease in TE cells, however it did not change the number of PE cells. In the second experiment, no embryo showed a fluorescent signal, so all blastocysts had their DNA extracted for genotyping. We sampled 34 embryos and one presented a band corresponding to the insertion site and also a band corresponding to the control embryo (wild type), suggesting mosaicism. We observed that best chances of gene insertion occurred when the injection of components of the CRISPR system was performed after 8 hours of IVF and with higher concentrations. In conclusion, we were able to remove SFB from our embryo production system and surprisingly SFB did not influence differentiation into PE. Still, further studies are needed in order to optimize the tool for obtaining knock-in in bovine embryos, mainly to obtain higher success rates and reduce the probability of occurrence of mosaicism in these embryos. Palavras-chave em inglês (separadas por vírgula): Gene editing. CRISPR/Cas9. Homologous Recombination. Blastocyst

Bovine conceptus of Bos indicus produced by somatic cell nuclear transfer and parthenogenesis present morphological variations since the blastocyst stage / Conceptos bovinos (Bos indicus) produzidos por transferência nuclear de células somáticas e partenogênese apresentam variações morfológicas desde o estágio de blastocisto

In cattle, embryo development is characterized by the appearance of two distinct cell layers, the trophectoderm and the inner cell mass. The latter will undergo differentiation to form the embryonic disc consisting of the epiblast and hypoblast. The aim of this study was to ultrastructurally characterize the bovine embryo from different in vitro production techniques, with emphasis on trophectoderm and inner cell mass cells. Bovine embryos on day 7 (conception = D1) of pregnancy, derived via in vitro production techniques, were fixed for light and transmission electron microscopy processing. Results suggested that embryos produced by nuclear transfer of somatic cells and parthenogenesis showed significant changes in macroscopic and microscopic structure. Size was reduced, and the inner cell mass had no defined shape. Furthermore, organelles responsible for the absorption processes, communication, growth, and cellular metabolism were fewer and had changes in shape, when compared to results in embryos produced by in vitrofertilization. We concluded that embryos produced by parthenogenesis and SCNT exhibit morphological differences when compared with IVF embryos, such as undeveloped blastocoel, poorly defined distribution of ICM, and morphological differences in organelles(AU)

Em bovinos, o desenvolvimento embrionário é caracterizado pelo surgimento de duas camadas distintas, o trofectoderma e a massa celular interna. Este último irá sofrer diferenciação para formar o disco embrionário, o qual consiste em epiblasto e hipoblasto. O objetivo deste estudo foi caracterizar ultraestruturalmente o embrião bovino proveniente de diferentes técnicas de produção in vitro, com ênfase no trofectoderma e na massa celular interna. Embriões bovinos com sete dias de gestação (fecundação = D1), derivados de técnicas de produção in vitro, foram fixados para processamento de microscopia de luz e eletrônica de transmissão. Os resultados sugerem que os embriões produzidos por transferência nuclear de células somáticas e partenogênese apresentaram alterações significativas em suas estruturas macro e microscópica. O tamanho foi reduzido, e a massa celular interna não tinha uma forma definida. Além disso, organelas responsáveis por processos de absorção, comunicação, crescimento e metabolismo celular estavam em menor número e tinham alterações na forma quando comparadas aos resultados em embriões produzidos por fertilização in vitro. Conclui-se que os embriões produzidos por SCNT e partenogênese apresentam diferenças morfológicas quando comparados aos embriões de fertilização in vitro, tais como blastocele pouco desenvolvida, massa celular interna pouco definida e diferenças morfológicas nas organelas(AU)

Influência da via de sinalização HIPPO na segregação entre a massa interna celular e o trofectoderma em embriões bovinos

Resumo: O primeiro evento de diferenciação celular em mamíferos consiste na separação entre a massa celular interna (MCI) e o trofectoderma (TE). Em camundongos, a via de sinalização HIPPO atua no controle da expressão de genes que definem essa diferenciação. Em bovinos, há indícios que os mesmos genes não participam da mesma maneira, porém a via HIPPO pode estar implicada nesse evento biológico. No presente estudo buscou-se testar a hipótese de que a atividade da proteína quinase LATS2 é necessária para a diferenciação da MCI em embriões bovinos. Para isso, realizou-se edição gênica via sistema CRISPR/Cas9 com intuito de inibir a função quinase de LATS2. Zigotos bovinos produzidos in vitro foram microinjetados com RNAs guia para o gene LATS2 acrescidos da enzima de restrição Cas9. Os zigotos foram separados em 3 grupos experimentais, sendo: grupo controle (não microinjetado), grupo Cas9 (microinjetado apenas com a proteína Cas9) e grupo gRNA (microinjetado com dois RNAs guias para o gene LATS2 e proteína Cas9). Os embriões foram cultivados para avaliação da taxa de formação de blastocistos, distribuição celular e fixados em paraformol 3,8% para posterior realização da imunofluorescência para YAP, seguido de avaliação de genótipo. Os resultados obtidos sugeriram que a clivagem não foi afetada no grupo editado, sendo as taxas 58%, 36% e 50% para grupo controle, Cas9 e gRNA respectivamente; porém, as taxas de formação de blastocisto sugeriram uma interferência direta no grupo editado, sendo 30%, 10% e 6% respectivamente, para os grupos controle, Cas9 e gRNA. Observou-se no âmbito morfológico que os embriões do grupo gRNA aparentavam estruturas assemelhadas a mórulas, sugestivo de morte embrionária ou bloqueio da diferenciação nessa fase do desenvolvimento. Houve redução numérica no número de células nas estruturas do grupo gRNA. As imagens obtidas via microscopia confocal, após a realização de imunofluorescência para YAP, demonstram a marcação de células do TE nos embriões microinjetados apenas com Cas9 e controle, porém, essa marcação não ocorreu nas no grupo gRNA em embriões que não formaram blastocele. Após a extração individual de DNA dos embriões testados na imunofluorescência, a PCR da região alvo de LATS2 resultou em uma banda de 478pb, além da banda original de 650pb, em uma amostra do grupo gRNA. Após sequenciamento dos produtos de PCR pelo método Sanger, constatou-se a alteração genética oriunda da deleção de trecho do gene LATS2 em 2 embriões, demonstrando o sucesso da edição gênica e corroborando com os demais achados. Isso sugere que a via HIPPO possui papel significativo na diferenciação de MCI e TE em embriões bovinos e de que o gene LATS2 está ligado à formação de blastocistos na espécie bovina.

Abstract: The first cellular differentiation event in mammals consists of the separation between the internal cellular mass (ICM) and the trophectoderm (TE). In mice, the HIPPO signaling pathway acts to control the expression of genes that define this differentiation. In cattle, there are indications that the same genes do not participate in the same way, but the HIPPO pathway may be involved in this biological event. In the present study we aimed to test the hypothesis that the activity of LATS2 kinase is necessary for the differentiation of ICM in bovine embryos. In order to achieve this, we employed CRISPR/Cas9 gene editing system to inhibit the kinase function of LATS2. In vitro produced bovine zygotes were microinjected with RNAs guide aiming the LATS2 gene plus Cas9 restriction enzyme. The zygotes were sorted in 3 experimental groups: control group (not microinjected), Cas9 group (microinjected only with Cas9 protein) and gRNA group (microinjected with guide RNAs aiming the LATS2 gene and Cas9 protein). The embryos were cultured to evaluate the rate of blastocysts formation, cellular distribution, then fixed in 3.8% paraformol for subsequent immunofluorescence for YAP, followed by genotyping. The results suggested that the cleavage was not affected in the edited group, as rates were 58%, 36% and 50% for control , Cas9 and gRNA groups respectively; however, the blastocyst formation rates suggested a direct interference in the edited group, being 30%, 10% and 6% respectively, for the control groups, Cas9 and gRNA. Morphologically, it was observed that the embryos of the gRNA group appeared as morulas, suggestive of embryonic death or inhibition of differentation in this phase of development. There was a numeric reduction in total cell count in the structures of the gRNA group.. The images obtained via confocal microscopy, after performing immunofluorescence for YAP demonstrate the marking of cells of the TE and MCI in the microinjected embryos only with Cas9 and control, however, this marking did not occur in the microinjected structures that did not form a blastocoel. After individual DNA extraction from the embryos tested in immunofluorescence, PCR of the LATS2 target region resulted in a band of 478pb, in addition to the original 650pb band, in one sample of the gRNA group. After Sanger sequencing of PCR products, genetic alteration derived from deletion of part of the LATS2 gene was observed in 2 embryos, demonstrating gene editing success and corroborating the findings. This suggests that HIPPO pathway has a significant role in the differentiation of MCI and TE in bovine embryos and that the LATS2 gene is linked to the formation of blastocysts in bovine species.

Biological roles of progesterone, prostaglandins, and interferon tau in endometrial function and conceptus elongation in ruminants

A large majority of pregnancy loss in cattle occus during the firt three weeks afeer conception particularly during the pperi-implantation period. This review integrantes established and new information on the biological role of ovarian progesterone (P4), prostaglandina (PGs) and interferon tau (IFNT) in endometrial function and conceptus elongation during the peri-implantation period of pregnancy in ruminants. Progesterone is secreted by the ovarian corpus luteum (CL) and is the u nequivocal hormone of pregnancy. Prostaglandins are produced from both the endometrium as well as conceptus trophectoderm during early pregnancy. Interferon tau is produced solely by the conceptus trophectoderm and is the maternal recognition of pregnancy signal that inhibits production of luteolytic pulses of PG F2α by the endometrium to maintain the CL and thus production of P4. Conceptus endometrial interactions in ruminants are complex and involve carefully orchestrated temporal and spatial alterations in endometrial gene expression during pregnancy. Available results support the idea that the individual, interactive, and coordinated actions of P4, PGs, and IFNT regulate uterine receptivity to conceptus implantation by controlling expression of genes in t he endometrium and that their actions are essential for conceptus elongation. One outcome of gene expression changes in the endometrial epithelia is alterations in lumenal secretions that govern conceptus elongation via effects on the trophectoderm. An increased knowledge of conceptus endometrial interactions during early pregnancy in ruminants is necessary to understand and elucidate the causes of infertility and recurrent pregnancy loss and to provide a basis for new strategies to improve fertility, pregnancy outcomes and thus reproductive efficiency.

Biological roles of progesterone, prostaglandins, and interferon tau in endometrial function and conceptus elongation in ruminants

A large majority of pregnancy loss in cattle occus during the firt three weeks afeer conception particularly during the pperi-implantation period. This review integrantes established and new information on the biological role of ovarian progesterone (P4), prostaglandina (PGs) and interferon tau (IFNT) in endometrial function and conceptus elongation during the peri-implantation period of pregnancy in ruminants. Progesterone is secreted by the ovarian corpus luteum (CL) and is the u nequivocal hormone of pregnancy. Prostaglandins are produced from both the endometrium as well as conceptus trophectoderm during early pregnancy. Interferon tau is produced solely by the conceptus trophectoderm and is the maternal recognition of pregnancy signal that inhibits production of luteolytic pulses of PG F2α by the endometrium to maintain the CL and thus production of P4. Conceptus endometrial interactions in ruminants are complex and involve carefully orchestrated temporal and spatial alterations in endometrial gene expression during pregnancy. Available results support the idea that the individual, interactive, and coordinated actions of P4, PGs, and IFNT regulate uterine receptivity to conceptus implantation by controlling expression of genes in t he endometrium and that their actions are essential for conceptus elongation. One outcome of gene expression changes in the endometrial epithelia is alterations in lumenal secretions that govern conceptus elongation via effects on the trophectoderm. An increased knowledge of conceptus endometrial interactions during early pregnancy in ruminants is necessary to understand and elucidate the causes of infertility and recurrent pregnancy loss and to provide a basis for new strategies to improve fertility, pregnancy outcomes and thus reproductive efficiency.(AU)

Uso de meio condicionado por células estromais mesenquimais uterinas durante o cultivo in vitro de embriões bovinos

As tecnologias de reprodução assistida, tais como a fertilização in vitro (FIV), transferência de embriões, transgenia e clonagem, ainda não tem o impacto comercial desejado devido a baixa produção embrionária. Apenas 30 a 40% dos blastocistos desenvolvidos são obtidos de oócitos após a MIV, fertilização e cultivo dos embriões, embora 80% dos oócitos maturados in vitro sejam fertilizados com sucesso. O soro fetal bovino (SFB) é o suplemento mais utilizado no cultivo de embriões in vitro, uma vez que melhora o desenvolvimento dos blastocistos. Apesar disso, sua presença está relacionada a alterações do metabolismo embrionário, perda de qualidade e indução de modificações na expressão de vários genes embrionários. Na tentativa de minimizar os efeitos deletérios do SFB, várias citocinas e fatores de crescimento têm sido acrescentados aos meios de cultivo embrionários in vitro, com a intenção de mimetizar as condições de cultivo in vivo. O presente experimento tem como objetivo avaliar e comparar os efeitos da adição do SFB e de meio condicionado por células mesenquimais estromais (MSCs) durante o cultivo embrionário. Os parâmetros analisados foram a viabilidade embrionária, apoptose e o perfil transcricional de genes relacionados à qualidade dos embriões. Não foi observado uma diferença (P0,05) na clivagem dos blastocistos, porém observou-se que a taxa de produção de embriões utilizando SFB no CIV foi maior (P0,05) quando comparada com MC, mas não diferiu (P0,05) do grupo produzido com BSA. A expressão relativa de mRNA para ELOVL6 foi maior no grupo MC, para CASP3 no grupo BSA, e para ACSL3 e VEGF no grupo SFB. Em conjunto, esses dados sugerem que o MC pode ser utilizado como uma alternativa ao SFB. Observamos um perfil de expressão gênica diferente, sugerindo que o MC inibiu o aumento da expressão relativa de mRNA para CASP3. Ademais, o MC favoreceu o número total de células, inibiu a porcentagem de células em apoptose e produziu um embrião de melhor qualidade.

Endometrial mesenchymal stromal cell (eMSCs) secretes bioactive molecules such as cytokines and growth factors which are released as soluble factors or through EVs. CM of the MSCs maintains the immunomodulation and regenerative potential properties of the cells that produced it. This study investigated the use of eMSC-CM plus BSA as an alternative to FBS in bovine embryo culture medium. The developmental ability and quality of bovine embryos were determined by assessing their cell number and gene expression. The percentage of embryos that underwent cleavage was similar (P>0.05) among the groups but blastocyst formation was higher (P<0.05) in FBS group. The total cell number was higher in CM group, but not statistically different from the others (P>0.05). The relative mRNA expression of ELOVL6 was higher in the CM group, of CASP3 in the BSA group and of ACSL3 and VEGF in the FBS group. Taken together, these data suggest that CM can be used as an alternative supplement to FBS. The gene expression profile suggested that the use of CM inhibited an increase in the relative mRNA levels for CASP3. Moreover, the CM favored the total number cells, inhibited the percentage of cells in apoptosis and produces better quality embryo.

Efeitos da substituição de SFB por IGF-I sobre os aspectos celulares e moleculares da produção in vitro de embriões bovinos

O objetivo do presente estudo foi avaliar o benefício da substituição do soro fetal bovino (SFB) pelo fator de crescimento semelhante à insulina tipo I (IGF-1) durante a maturação in vitro (MIV) ou cultivo in vitro (CIV), sobre qualidade embrionária e expressão de genes em embriões pré- e pós-compactação. Delineamento experimental foi fatorial 3 x 3 (três suplementos de MIV e três de CIV), com 9 grupos experimentais. Foram realizadas 20 réplicas (oócitos 400/grupo). Complexos cúmulus-oócito (COCs) graus I e II foram maturados in vitro com a adição de 10% de SFB (SFB), ou 3 mg/mL polivinil-álcool (PVA), ou PVA + 100 ng/mL de IGF-1 (IGF) a 38,5 ºC em 5% de CO2 em ar por 22 a 24 horas. Foram fertilizados e incubados durante 18 horas. Os zigotos foram cultivados com a adição de: 2,5% de SFB (SFB), ou 3 mg/mL de PVA (PVA), ou PVA + 100 ng/mL de IGF-1 (IGF) por sete dias a 38,5ºC em 5% de CO2 em ar. As taxas de clivagem e blastocisto foram avaliadas após 48 e 168 horas de cultivo, respectivamente. A técnica simplificada de coloração diferencial de células da massa celular interna (MCI) e trofoectoderma (TE) foi utilizada para avaliar a distribuição celular de blastocistos (n = 155) e a técnica de TUNEL para avaliação do índice de apoptose (n = 207). Concentrações de glicose e lactato obtidas do meio MIV utilizaram-se para analisar o metabolismo de glicose. mRNA foi extraído de embriões de 6 8 células colhidos após 66 horas post-inseminação (4 pools de 15 embriões por grupo) e de blastocistos expandidos de 7 dias (4 pools de 5 embriões por grupo). A expressão gênica foi realizada no sistema BioMark HD® de microfluídica, pelo arranjo 96.96 Dynamic Array. Os dados foram analisados ANOVA do PROC GLIMMIX do SAS. Foi utilizado o teste Tukey para comparação de médias. Valores de p 0.05 foram considerados significativos. A clivagem foi maior (p < 0,05) nos grupos maturados em SFB. MIV e CIV com SFB presentou maior (p < 0.05) taxa de produção de blastocisto e maior quantidade de blastocistos expandidos que PVA e IGF. MIV com IGF-I aumentou o consumo de glicose e síntese de lactato dos COCs e levou à produção de blastocistos com maior (p < 0.05) número total de células. Embriões cultivados em IGF-I tiveram maior (p < 0.05) quantidade de células na MCI e embriões cultivados em PVA tiveram maior (p < 0.05) apoptose do que SFB. Os genes NANOG, OTX2, POU5F1 e IFNT2 foram mais expressos em blastocistos cultivados em PVA e IGF do que em SFB. O IGF-I regula TP53, BAX, CASP3, CASP9, HSPA1A e IGF1R para prevenir apoptose. IGF-I durante a MIV em meio semi-definido estimula o metabolismo de glicose de COCs, melhora a qualidade embrionária, aumentando o número total de células de blastocistos. IGF-I durante a CIV aumenta o numero de células na MCI, melhora a expressão de biomarcadores importantes de qualidade embrionária com a possibilidade de melhorar o desenvolvimento embrionário.

The aim of the present study was to analyze the benefits of fetal bovine serum (FBS) replacement by insulin-like growth factor I (IGF-I) during in vitro maturation (IVM) and in vitro culture (IVC), on embryo quality and temporal gene expression in embryos pre- and post-compaction. A 3 x 3 factorial design was performed (three supplements for IVM and three for IVC), with a total of 9 experimental groups. A total of 20 replicates (oocytes 400/group) were performed. Grade I and II cumulus-oocyte complexes (COCs) matured in vitro with the addition of 10% of FBS (FBS), or 3 mg/mL of polyvinyl-alcohol (PVA), or PVA + 100 ng/mL IGF-1 (IGF) at 38.5 ºC in an atmosphere of 5% CO2 for 22 to 24 hours. After IVM, oocytes were fertilized and incubated for 18 hours. Possible zygotes were cultured with the respective addition of: 2.5% of FBS (FBS), or 3 mg/mL of PVA (PVA), or PVA + 100 ng/mL of IGF-1 (IGF) for seven days at 38.5 ºC in an atmosphere of 5% CO2. Cleavage and blastocyst rates were analyzed at 48 and 168 hours of culture respectively. Simplified technique for differential staining of inner cell mass (ICM) and trophectoderm (TE) cells was performed to analyze cell allocation of blastocysts (n = 155) and TUNEL assay for apoptosis rate analysis (n = 207). Glucose and lactate concentrations were measured in IVM spent media to analyze glucose metabolism. mRNA was extracted from 6 8 cells embryos collected after 66 hours post insemination (4 pools of 15 embryos per group) and 7 day expanded blastocysts (4 pools of 5 embryos per group). Gene expression analysis was performed with BioMark HD® system with microfluidic chip 96.96 Dynamic Array. Data were analyzed by ANOVA from PROC GLIMMIX model from SAS. Tuckey test was used to compare means. P value 0.05 was considered to be significant. Cleavage rate was higher (p < 0.05) for groups matured in FBS. IVM and IVC in FBS presented higher (p < 0.05) total blastocyst yield and greater quantity of expanded blastocysts than PVA and IGF. IVM with IGF-I increased glucose uptake and lactate synthesis of COCs and produced blastocysts with increased (p < 0.05) total cell number. Embryos cultured in IGF-I had greater (p < 0.05) amount of cells in the ICM and embryos cultured in PVA had higher (p < 0.05) apoptosis rate than FBS. NANOG, OTX2, POU5F1 and IFNT2 genes were more expressed in blastocysts cultured in PVA and IGF than in FBS. IGF-I regulates TP53, BAX, CASP3, CASP9, HSPA1A and IGF1R genes to prevent apoptosis. The addition of IGF-I during IVM in chemically semi-defined media stimulates glucose metabolism of COCs and improves embryo quality, increasing total cell number of blastocysts. The addition of IGF-I during IVC increases the amount of cells of the ICM, improves expression of important embryo quality biomarkers with the possibility to enhance embryo development.

Uterine environment and conceptus development in ruminants

Interferon tau (IFNT), the pregnancy recognition signal from trophectoderm cells of ruminant conceptuses abrogates the uterine luteolytic mechanism to ensure maintenance of functional corpora lutea for production of progesterone (P4). IFNT acts in concert with P4 to induce expression of genes for transport and/or secretion of histotroph that includes nutrients such as glucose and arginine that activate the mechanistic target of rapamycin (MTOR) nutrient sensing cell signaling pathway to stimulate proliferation, migration, differentiation and translation of mRNAs essential for growth and development of the conceptus. Arginine, leucine, glutamine and glucose increase in the uterine lumen during the peri-implantation period of pregnancy due to increased expression of their transporters by uterine luminal epithelium (LE) and superficial glandular epithelium (sGE) in response to P4 and IFNT. In day 16 ovine conceptus explant cultures, arginine increases GTP cyclohydrolase 1 mRNA, and IFNT, while arginine and glucose increase ornithine decarboxylase, nitric oxide synthase 2, and GCH1. Arginine can be metabolized to nitric oxide (NO) and polyamines which stimulate proliferation of ovine trophectoderm (oTr) cells. Secreted phosphoprotein 1 (SPP1, also known as osteopontin) in uterine histotroph increases focal adhesion assembly as a prerequisite for adhesion and migration of oTr cells through activation and cross-talk between MTOR, MAPK, and myosin II motor pathways. Glucose, arginine, leucine and glutamine stimulate MTOR signaling, proliferation and mRNA translation by oTr cells. Further, glucose and fructose were equivalent in stimulating proliferation and synthesis of hyaluronic acid via the hexosamine pathway in oTr and pig Tr cells. These mechanisms allow select nutrients and SPP1 to act coordinately to affect synthesis of proteins involved in cell signaling affecting conceptus growth, development, and survival during the peri-implantation period of pregnancy.

Uterine environment and conceptus development in ruminants

Interferon tau (IFNT), the pregnancy recognition signal from trophectoderm cells of ruminant conceptuses abrogates the uterine luteolytic mechanism to ensure maintenance of functional corpora lutea for production of progesterone (P4). IFNT acts in concert with P4 to induce expression of genes for transport and/or secretion of histotroph that includes nutrients such as glucose and arginine that activate the mechanistic target of rapamycin (MTOR) nutrient sensing cell signaling pathway to stimulate proliferation, migration, differentiation and translation of mRNAs essential for growth and development of the conceptus. Arginine, leucine, glutamine and glucose increase in the uterine lumen during the peri-implantation period of pregnancy due to increased expression of their transporters by uterine luminal epithelium (LE) and superficial glandular epithelium (sGE) in response to P4 and IFNT. In day 16 ovine conceptus explant cultures, arginine increases GTP cyclohydrolase 1 mRNA, and IFNT, while arginine and glucose increase ornithine decarboxylase, nitric oxide synthase 2, and GCH1. Arginine can be metabolized to nitric oxide (NO) and polyamines which stimulate proliferation of ovine trophectoderm (oTr) cells. Secreted phosphoprotein 1 (SPP1, also known as osteopontin) in uterine histotroph increases focal adhesion assembly as a prerequisite for adhesion and migration of oTr cells through activation and cross-talk between MTOR, MAPK, and myosin II motor pathways. Glucose, arginine, leucine and glutamine stimulate MTOR signaling, proliferation and mRNA translation by oTr cells. Further, glucose and fructose were equivalent in stimulating proliferation and synthesis of hyaluronic acid via the hexosamine pathway in oTr and pig Tr cells. These mechanisms allow select nutrients and SPP1 to act coordinately to affect synthesis of proteins involved in cell signaling affecting conceptus growth, development, and survival during the peri-implantation period of pregnancy.(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)