Maternal contributions from the uterine endometrial luminal (LE) and glandular (GE) epithelia are unequivocally required to support ruminant conceptus growth and development, elongation and implantation. Therefore, studies were conducted to examine expression of endometrial genes hypothesized to regulate conceptus development. The first study investigated two genes specifically expressed in the LE and superficial GE of the ovine uterus. Insulin-like growth factor binding protein (IGFBP1) and (IGFBP3) expression was coordinate with ovine conceptus elongation. Treatment with P4 induced and IFNT stimulated IGFBP1, but not IGFBP3; however, IFNT only moderately stimulated IGFBP1, indicating that another conceptus-derived factor stimulates endometrial IGFBP1 expression. IGFBP1 did not affect proliferation of ovine trophectoderm (oTr) cells in vitro, but stimulated their migration and attachment. Results indicated that IGFBP1, but not IGFBP3 is a marker of conceptus elongation in ruminants and stimulates cell migration and attachment. The second study evaluated the effects of pregnancy, P4 and IFNT on expression of hydroxysteroid (11-beta) dehydrogenases (HSD11B1 and HSD11B2), nuclear receptor subfamily 3, group C, member 1 (NR3C1), and prostaglandin-endoperoxide synthase 2 (PTGS2) in the ovine uterus. Expression of HSD11B1 mRNA and PTGS2 protein in endometrial LE and sGE were coordinate with conceptus elongation, while HSD11B2 mRNA was expressed primarily in the conceptus. Further, P4 induced, but IFNT only moderately stimulated HSD11B1. Thus, HSD11B1 expression may be regulated by prostaglandins (PGs) during early pregnancy. The presence of NR3C1 in the ovine uterus implicates cortisol, the main product of HSD11B1, in peri-implantation period events that include elongation of the ovine conceptus. The third study determined in vivo effects of PGs on ovine conceptus elongation and endometrial gene expression. Compared to control ewes, intrauterine infusions of a PTGS2 inhibitor, meloxicam, retarded elongation and decreased expression of elongation-related genes including IGFBP1, IGFBP3, HSD11B1, galectin 15 (LGALS15), solute carrier family 2, member 1 (SLC2A1), gastrin-releasing peptide (GRP), cystatin C (CST3), radical S-adenosyl methionine domain containing 2 (RSAD2), and ISG15 ubiquitin-like modifer (ISG15). Collectively, these studies assessed the effects of pregnancy, P4, IFNT, and PGs on endometrial genes implicated in conceptus growth. These results indicate that IGFBP1 is a marker of conceptus elongation in ruminants and provide novel roles for both cortisol and PGs in endometrial gene expression and conceptus elongation.

This research was conducted to test the hypothesis that select nutrients including glucose, leucine, arginine and glutamine stimulate conceptus development by activating mTOR (mammalian target of rapamycin; HGNC approved gene name: FRAP1, FK506 binding protein 12-rapamycin associated protein 1) signaling pathway. First, temporal changes in quantities of select nutrients (glucose, amino acids, glutathione, calcium, sodium and potassium) in uterine lumenal fluid from cyclic (Days 3 to 16) and pregnant (Days 10 to 16) ewes were determined. Total recoverable glucose, Arg, Gln, Leu, Asp, Glu, Asn, His, beta-Ala, Tyr, Trp, Met, Val, Phe, Ile, Lys, Cys, Pro, glutathione, calcium and sodium was greater in uterine fluid of pregnant compared to cyclic ewes between Days 10 and 16 after onset of estrus. Of note were remarkable increases in glucose, Arg, Leu and Gln in uterine flushings of pregnant ewes between Days 10 and 16 of pregnancy. Second, effects of the estrous cycle, pregnancy, progesterone (P4) and interferon tau (IFNT) on expression of both facilitative (SLC2A1, SLC2A3 and SLC2A4) and sodium-dependent (SLC5A1 and SLC5A11) glucose transporters, cationic amino acid transporters (SLC7A1, SLC7A2 and SLC7A3), neutral amino acid transporters (SLC1A4, SLC1A5, SLC3A1, SLC6A14, SLC6A19, SLC7A5, SLC7A6, SLC7A8, SLC38A3, SLC38A6 and SLC43A2) and acidic amino acid transporters (SLC1A1, SLC1A2 and SLC1A3) in ovine uterine endometria from Days 10 to 16 of the estrous cycle and Days 10 to 20 of pregnancy as well as in conceptuses from Days 13 to 18 of pregnancy were determined. Among these genes, SLC2A3 and SLC7A6 were detectable only in trophectoderm and endoderm of conceptuses. The abundance of mRNAs for SLC2A1, SLC2A4, SLC5A1, SLC5A11, SLC7A1, SLC7A2, SLC1A4, SLC1A5, SLC43A2 and SLC1A3 changed dynamically in ovine uterine endometria according to day of the estrous cycle and early pregnancy. Expression of mRNAs for SLC2A1, SLC5A11 and SLC7A1 in endometria was induced by P4 and further stimulated by IFNT with shortterm treatment (12 days), while expression of SLC7A1 and SLC1A5 in endometria required long-term treatment (20 days) with P4 and IFNT. Third, effects of the estrous cycle, pregnancy, P4 and IFNT on expression of nitric oxide synthase (NOS1, NOS2 and NOS3), GTP cyclohydrolase (GCH1), ornithine decarboxylase 1(ODC1), insulin-like growth factor II (IGF2), FRAP1 complexes (FRAP1, LST8, MAPKAP1, RAPTOR, RICTOR), regulators (TSC1, TSC2, RHEB) and an effector (EIF4EBP1) of FRAP1 signaling in ovine uterine endometria from Days 10 to 16 of the estrous cycle and Days 10 to 20 of pregnancy as well as in conceptuses from Days 13 to 18 of pregnancy were determined. All of these genes were expressed in ovine uterine endometrium and conceptuses. Among these genes, expression of NOS1, IGF2, RHEB and EIF4EBP1 changed dynamically due to day of the estrous cycle and early pregnancy. Progesterone stimulated NOS1 and GCH1 expression while IFNT inhibited NOS1 expression in uterine endometria, and P4 and IFNT stimulated expression of RHEB and EIF4EBP1 in uterine endometria. Collectively, these results indicate that: 1) the availability of select nutrients in the ovine uterine lumen increases to support the rapid growth and elongation of the conceptus during the peri-implantation stage of pregnancy; 2) P4 and/or IFNT stimulate(s) glucose and amino acid transporters to facilitate their transport from maternal tissues and/or blood into the uterine lumen during early pregnancy; 3) the FRAP1 cell signaling pathway mediates interactions between the maternal uterus and peri-implantation conceptus and both P4 and IFNT affect this pathway by regulating expression of RHEB and EIF4EBP1. Expression of NOS, ODC1 and IGF2 appear to be linked to FRAP1 signaling in both uteri and peri-implantation conceptuses.

Galectins are a family of secreted animal lectins with a high affinity to betagalactosides commonly involved in cellular functions such as apoptosis, adhesion and migration. Galectin 15 (LGALS15), a newest member of the galectin superfamily, has a unique C-terminal RGD sequence and participates in integrin-mediated ovine trophectoderm cell attachment and migration. In the ovine uterus, LGALS15 is expressed only by the endometrial luminal (LE) and superficial glandular (sGE) epithelia, induced by progesterone between Days 10 and 12 of the cycle and pregnancy, and then stimulated by interferon tau (IFNT) from the conceptus after Day 14 of pregnancy. During early pregnancy, the canonical janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway is not active in the endometrial LE/sGE. Therefore, IFNT may utilizes a non-canonical signaling pathway to increase transcription of genes, including CST3, CTSL, HIF2A, LGALS15, and WNT7A, specifically in the endometrial LE/sGE. Alternatively, IFNT and progesterone could indirectly affect epithelial gene expression by influencing gene expression in the stroma, which then communicates with the epithelium. Although the LGALS15 gene is present in ovine, caprine and bovine species, it is only expressed in uteri of sheep and goats. Available data shows a tissue- and speciesspecific expression pattern for LGALS15, likely involving multiple layers of transcription regulation in the ruminant endometrium. Further analysis of the LGALS15 5? promoter/enhancer region revealed similar predicted transcription factor binding sites in all three species, including; PU.1, Ets-1, AP1, Sp1, and GRE or PRE sites. Interestingly, the proximal promoter region of the LGALS15 gene in all three species exhibited a conserved Sp1 binding site upstream of an AP1 binding site on both sense and antisense strands, and with similar spacing between binding sites. Sequence analysis revealed key differences in LGALS15 gene structure between ruminant species including the proximity of repetitive DNA sequences to the transcription start site (+1). Bovine LGALS15 has repetitive DNA sequences start at - 145 whereas in ovine or caprine LGALS15 it starts at about -300. The length of the repetitive DNA sequence is similar (~1.2 kb) in the 5' promoter/enhancer region of LGALS15 in all three species. Transient transfection analyses found that repetitive DNA sequences reduced basal promoter activity and responsiveness to treatments. None of the promoter construct showed responsiveness to interferon tau (IFNT). The bovine LGALS15 gene promoter showed no activity under any experimental conditions. The current studies indicate that uterine LGALS15 is expressed in ovine and caprine but not bovine species. Additionally, repetitive DNA sequences found in the promoter region may contribute to modulating the LGALS15 gene expression. Therefore, the ruminant LGALS15 gene, like other galectins, is under tight transcriptional control involving hormones, requisite transcription factors and potentially chromatin remodeling complexes working synergistically for LGALS15 promoter transactivation.

In vitro systems like those used for in vitro embryo production are invaluable for our understanding of embryogenesis and the processes that regulate it. However, extensive research has also highlighted that in vitro produced embryos negatively differ from their in vivo counterparts in various ways. Not surprisingly, there is ~20% decrease in pregnancy success from pregnancies established using in vitro produced embryos. Therefore, much research has relied on attempting to produce a better in vitro embryo that more closely resembles their in vivo counterparts. Our laboratory has investigated this by supplementing a cytokine, interleukin-6 (IL6), during in vitro embryo culture. My dissertation work expands upon those initial efforts by answering more detailed questions related to the biological role of IL6 during cattle embryogenesis. In the work presented herein, IL6 supplementation during in vitro culture was able to transform the transcriptome of resulting conceptuses post embryo transfer. The transcriptome of these conceptuses included an abundance of genes associated with survival. Indeed, we witnessed IL6-treated conceptuses resulted in a 20% increased survival rate and were longer than their non-treated counterparts. In the second research project, we employed CRISPR-Cas9 genome editing technology to understand the embryo phenotype after part of the IL6 receptor responsible for signal transduction, interleukin-6 signal transducer (IL6ST), is disrupted. We discovered that IL6ST is required for development before the blastocyst stage. In addition, IL6ST disrupted blastocysts, presumed to contain wildtype, presented with severe, abnormal morphology. Not only did this group of embryos have decreased ICM and TE cell numbers, but they also had an increased occurrence of cells within the TE region that were negative for its traditional marker, CDX2. This suggests IL6ST is likely involved in a pathway responsible for determining cell fate identity at the blastocyst stage. Collectively, IL6 in cooperation with IL6ST, is a key controller of embryogenesis in cattle. / Doctor of Philosophy / There are major events that an embryo must successfully advance from to continue development to form into an organism capable of survival after birth. Over 30% of pregnancies in cattle and humans will fail within the first 30 days of gestation. This time period coincides with several key developmental events that ultimately modify the morphology of the growing embryo. Our laboratory primarily focuses on embryo development around the blastocyst stage. If an embryo advances to this stage, it has a greater likelihood of maintaining viability. Therefore, my dissertation research has focused on early embryonic development from the time of first cleavage (~day 2 of gestation) through embryo elongation (~day 15 of gestation), which encompasses the blastocyst stage. Within this time frame, I have been investigating embryonic effects after supplementation of a protein, interleukin-6 (IL6). Previously, our laboratory has identified IL6 to cause favorable impacts on the developing embryo, but its mode of action was unknown. Therefore, my dissertation research has investigated the mechanistic actions of IL6, and its beta receptor subunit, interleukin-6 signal transducer (IL6ST). In my first research project, we discovered that supplementing IL6 during in vitro embryo culture resulted in increased embryo elongation and survival. In my second research project, we found IL6ST is an absolute requirement for embryo survival to the blastocyst stage. Together, these results indicate IL6 is a very important protein needed for sustained pregnancy viability.

This dissertation examines the protection given by the Civil Code and the Constitution to the different stages of human life, considering since the embryos produced in vitro until the moment of birth. The problem is analyzed from a fundamental rights perspective, contrasting Bioethics and Biolaw with Constitutional Law, as well as the civil concepts of legal subjects and legal personality. This dissertation considers the embryo and the conceptus as a subject of law, without including them in the juridical concept of person. Thus, the human life still in development receives legal protection, but the law does not specify clearly the limits of this protection, especially if they would be under the legal protection of life. The new Biosafety Act relativized the legal protection of human life in embryonic stage, enabling research with embryos resulting from assisted reproduction. The Criminal Code also relativizes the juridical protection of life, allowing the abortion under certain circumstances. This definition is important so that biojuridical issues receive a just legal protection. Considering the absence of a statutory definition, this dissertation analyzes, from a constitutional hermeneutics perspective, the possibility to ponder the right to life with other fundamental rights, especially the right to health, the right to genetic heritage and reproductive rights. This dissertation suggests it is possible to limit the protection of life of these subjects in contrast with the protection of other human rights guaranteed to the person already born. The principle of the dignity of human life in relation to the principle of human dignity is used as an hermeneutical parameter. With this analysis, this dissertation examines the decisions of the Supreme Court until June 2011, in which the terms “embryo” and “conceptus” are analyzed to consider the attitude adopted by the Ministers facing these statutory omissions. / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / O presente trabalho tem por objeto a análise da proteção civil-constitucional conferida ao ser humano quando ainda em formação gestacional, considerando desde o embrião laboratorial até o momento do nascimento. A problemática é analisada sob a perspectiva dos direitos fundamentais, na interface da Bioética e do Biodireito com o Direito Constitucional, além dos institutos do Direito Civil de sujeito de direito e da personalidade jurídica. Entende-se o embrião e o nascituro enquanto sujeito de direito, sem, contudo, serem considerados pessoas. Com isso, o ser humano ainda em formação recebe proteção jurídica, mas o Direito não especifica claramente qual o limite dessa proteção, principalmente se estariam resguardados pelo direito à vida. A nova Lei da Biossegurança relativizou a proteção à vida humana em formação, possibilitando as pesquisas que se utilizam do embrião humano laboratorial; o Código Penal, em certas circunstâncias, também relativiza a proteção à vida do nascituro, possibilitando o aborto. Essa definição é importante para que as questões biojurídicas recebam uma proteção jurídica adequada. Ante a ausência de uma norma definidora, o trabalho analisa, sob a ótica da hermenêutica constitucional, a possibilidade de ponderar o direito à vida em face de outros direitos fundamentais assegurados, principalmente o direito à saúde, o direito ao patrimônio genético e o direito sexual reprodutivo. Com isso, entende-se ser possível limitar a proteção à vida desses sujeitos em face da proteção de outros direitos assegurados à pessoa humana já nascida. Utiliza-se como parâmetro hermenêutico o princípio da dignidade da vida humana em relação ao princípio da dignidade da pessoa humana. Após essa análise doutrinária acerca do tema proposto, estudam-se os julgados do Supremo Tribunal Federal até junho de 2011, cujos termos “embrião” e “nascituro” surgem como objeto de apreciação para se constatar a postura adotada pelos Ministros diante dessas omissões legais.