ELUCIDATION OF THE ROLE OF AGOUTI-SIGNALING PROTEIN THROUGHOUT FOLLICULOGENESIS AND EARLY EMBRYONIC DEVELOPMENT IN CATTLE

The oocyte expresses certain genes during folliculogenesis to regulate the acquisition of oocyte competence. Oocyte competence, which refers to the presence of imperative molecular factors in the oocyte that are critical for high oocyte quality, is directly related to the ability of the oocyte to result in a successful pregnancy following fertilization. Over the past few decades, the development and optimization of assisted reproductive technologies, particularly in vitrofertilization, have enabled the beef and dairy industries to advance cattle genetics and productivity. However, only approximately 40% of bovine embryos will develop to the blastocyst stage in vitro. In addition, bovine embryos produced in vitro are developmentally inferior compared to in vivo derived embryos due to the lack of optimization of the oocyte and embryo culture conditions in vitro. Characterization of factors regulating these processes is crucial to improve the efficiency of bovine in vitro embryo production. RNA Sequencing data obtained by our laboratory demonstrated that the secreted protein, agouti-signaling protein (ASIP), is highly abundant in the bovine oocyte. Agouti-signaling protein (ASIP) has a characterized role in the distribution of melanin pigment in some mammalian species, including mice. In adipose tissue, ASIP expression is associated with insulin resistance and obesity. Recently, it was demonstrated that ASIP is crucial in regulating mammary epithelial cell lipid metabolism in cattle. However, the role of ASIP in the bovine oocyte and early embryo has not been previously elucidated. This research aimed to characterize the ASIP spatiotemporal expression profile in the ovary and throughout early embryonic development. Further, objectives included revealing the effects of supplementation of ASIP during in vitro oocyte maturation and embryo culture on subsequent embryonic development. In addition to oocyte expression, ASIP was detected in granulosa, cumulus, and theca cells isolated from antral follicles. Both ASIP mRNA and protein were found to decline with oocyte maturation, suggesting a prospective role for ASIP in achieving oocyte competence. Microinjection of presumptive zygotes using small interfering RNAs targeting ASIP led to a 13% reduction in the rate of development to the blastocyst stage. Additionally, we examined potential ASIP signaling mechanisms through which ASIP may function to establish oocyte developmental competence. Expression of melanocortin receptors 3 and 4 and the coreceptor attractin was detected in the oocyte and follicular cells. Interestingly, the addition of cortisol, which was previously determined to be beneficial for oocyte competence in cattle, during in vitro maturation significantly increased oocyte ASIP levels. Cumulus-oocyte complexes or presumptive zygotes were placed in culture medium containing either 0, 1, 10, or 100 ng/mL of recombinant ASIP, and effects on subsequent development, gene expression, lipid content, and blastocyst cell allocation were examined. Supplementation of ASIP during oocyte maturation improved the blastocyst development rate and produced blastocysts with an increased inner cell mass to trophectoderm cell ratio. Nile red staining revealed that adding ASIP during oocyte maturation increased oocyte but not embryo lipid levels. The expression of genes involved in lipid metabolism, including FASN, PPAR, SCD, CSL1, ELOVL5, and ELOVL6, were not found to be significantly altered in blastocysts due to treatment. Meanwhile, supplementation of ASIP during embryo culture did not affect blastocyst rates. These results support a functional role for ASIP in promoting oocyte maturation and subsequent embryonic development, potentially through signaling mechanisms involving cortisol. Additionally, these data further support the role of ASIP in acquiring oocyte competence and suggest that supplementing ASIP during oocyte maturation may lead to the production of blastocysts of increased quality. Future prospective applications of this work include optimizing bovine oocyte or embryo culture conditions to emulate better the in vivo maternal environment through normalizing lipid metabolism and, subsequently, minimizing stress. Further, future research should explore the utilization of ASIP in developing improved cryopreservation techniques for bovine embryos

In Vitro Embryo Production in Ruminants

This book presents the latest advances in assisted reproductive technologies applied to ruminants, such as multiovulation, artificial insemination, embryo transfer, and in vitro fertilization (IVF), which have been useful tools to accelerate the genetic progress in these species. The reader will find detailed studies on bisons, bovines, and goat

Análisis funcional del efecto de la nobiletina en el desarrollo embrionario bovino preimplantacional

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Veterinaria, Departamento de Bioquímica y Biología Molecular, leída el 16-07-2021In vitro production of embryos (IVP) is a reproductive biotechnology widely used to increase the number of offspring from superior phenotypes, to treat infertility problems and also to address fundamental questions about metabolic pathways that modulate early embryonic development. However, IVP is a multifactorial process depending on extrinsic and intrinsic factors, at both cellular and molecular levels, to increase its efficiency. Still today the IVP procedure has limitations, considering that not all oocytes have the ability to reach the blastocyst. Although a successful in vitro maturation (IVM) involves a nuclear, cytoplasmic and molecular maturation, necessary for fertilization and further embryo development, the blastocyst rate do not overcome 30-40% in bovine. Thus, alteration in the dynamics of early embryo development, which coincides with the switch from maternal control to embryonic genome activation (EGA) on Day 4 post fertilization, may be partially responsible for reduced embryo yield. Furthermore, it has been suggested that apart from the origin of the oocyte, the increase in reactive oxygen species (ROS) and the culture environment affecting EGA, there is also a complex network of signaling pathways responsible for cell division and differentiation and other events necessary for embryonic development, such as MAPK and PI3K/AKT...La producción in vitro de embriones (IVP) es una biotecnología reproductiva ampliamente utilizada para aumentar el número de descendientes de fenotipos superiores, para tratar problemas de infertilidad y también para abordar preguntas fundamentales sobre las vías metabólicas que modulan el desarrollo embrionario temprano. Sin embargo, la IVP es un proceso multifactorial que depende de factores extrínsecos e intrínsecos, tanto a nivel celular como molecular, para aumentar su eficacia. Aún hoy, la IVP tiene limitaciones, considerando que no todos los ovocitos tienen la capacidad de llegar a la etapa de blastocisto. Aunque una maduración in vitro (IVM) exitosa implica una maduración nuclear, citoplasmática y molecular, necesaria para la fecundación y el desarrollo posterior del embrión, la tasa de blastocistos no supera el 30-40% en bovinos. Por lo tanto, la alteración en la dinámica del desarrollo embrionario temprano, que coincide con el cambio del control materno a la activación del genoma embrionario (EGA) en el día 4 posterior a la fecundación, puede ser parcialmente responsable de la reducción del rendimiento embrionario. Se ha sugerido que además del origen del ovocito, el aumento de especies reactivas de oxígeno (ROS) y el ambiente de cultivo que afecta a EGA, existe una compleja red de vías de señalización responsables de la división y diferenciación celular y otros eventos necesarios para el desarrollo embrionario, como las cascadas genéticas MAPK y PI3K/AKT...Fac. de VeterinariaTRUEunpu

Non-Invasive Molecular Assessment to Determine Embryonic Viability and Reproductive Success

The success of human IVF is essentially determined by the quality of the embryo (itself a function of the quality of the oocyte and sperm), the endometrium and of the interaction between them. While there are established means of establishing the quality of each individually, some of these are limited and, in particular, the embryo-endometrial dialogue is difficult to capture and observe. As a result, it is under-studied and, with this in mind, the aims of this thesis were threefold: First, to investigate the corona cell transcriptome of euploid oocytes, RNA sequencing of corona cells from individual cumulus oocyte complexes that developed into euploid blastocysts was employed. Alongside bioinformatic and statistical analysis to compare IVF outcomes, a mean number of sequence reads of 21.2 million were produced. Differentially expressed gene analysis revealed 343 statistically significant transcripts and enriched pathway analysis showed WNT signaling, MAPK signaling, focal adhesion and TCA cycle to be related to IVF outcome. Specifically, key genes within the WNT/beta-catenin signaling pathway, including AXIN, were associated with oocyte competence. Furthermore, key genes and signaling pathways were identified in corona cell profiles in association with IVF outcome following the transfer of euploid blastocysts previously vitrified in a frozen embryo transfer. A second study investigated an association between advanced maternal age (AMA) and endometriosis on the embryo-endometrial molecular dialogue before implantation. Co-culture experiments were performed with endometrial epithelial cells (EEC) and cryopreserved day 5 blastocysts from AMA or endometriosis patients. Extracellular vesicles isolated from the co-culture supernatant were analyzed for miRNA expression and revealed significant alterations correlating to AMA or endometriosis. Functional annotation analysis of miRNA-target genes revealed enriched pathways for both infertility etiologies, including disrupted cell cycle regulation and proliferation. These extracellular vesicle-bound secreted miRNAs are key transcriptional regulators in embryo-endometrial dialogue. The third study was concerned with the placental epigenome, which plays a critical role in regulating mammalian growth and development. Alterations to placental methylation, often observed at imprinted genes, can lead to adverse pregnancy complications such as intrauterine growth restriction and preterm birth. Similar associations have been observed in offspring derived from advanced paternal age fathers. As parental age at time of conception continues to rise, the impact of advanced paternal age on these reproductive outcomes is a growing concern, but limited information is available on the molecular mechanisms affected in utero. This longitudinal murine research study investigated the impact of paternal aging on genomic imprinting. Paternal age significantly impacted embryonic placental weight, fetal weight and length, significant hypermethylation was observed upon natural paternal aging and several transcript level alterations attributable to advanced paternal age were identified. This thesis also presents a series of co-authored studies on related subjects such as embryonic epigenetic dysregulation, unexplained male factor and the sperm epigenome, the dynamic transcriptome of IVF blastocysts in association with infertility, redox balance and aging-related changes in the mouse ovary/oocyte, obesity and the human sperm proteome. Collectively, these data could inform the development novel biomarkers for non-invasive assessment of oocyte competence and implantation success. They also demonstrate a paternal age effect with dysregulation at numerous imprinted loci, providing a mechanism for future adverse placental and offspring health conditions

Exosomal and non-exosomal circulatory miRNAs in bovine follicular fluid : potential role of exosomal miRNAs in oocyte development

Growth and development of bovine follicle and oocyte is the result of series of complex and coordinated processes that involves extensive cell-to-cell communication in the follicle. This phenomenon involves enormous complex and heterogeneous biochemical substances existing in the oocytes and its surrounding cells and the follicular fluid. Cell-cell communication within the follicle involves many signaling molecules, and this process may be mediated by secretion and uptake of exosomes that contain several bioactive molecules including extra-cellular miRNAs. The molecular mechanism of oocytes development and interaction between oocyte and follicular cells in the follicular micro-environment remains vague. Follicular fluid and cells from individual follicles of cattle were grouped based on Brilliant Cresyl Blue Pro staining of the corresponding oocytes. Both ExoquickTM precipitation and differential ultracentrifugation were used to separate the exosome and non-exosomal portion of follicular fluid. Following miRNA isolation from both fractions, the human miRCURY LNATM Universal RT miRNA PCR array system was used to profile miRNA expression. Western blot analysis against specific protein and electron microscopy imaging confirms the efficient separation of exosomal and non-exosomal fraction of follicular fluid. The real time qPCR array analysis revealed that a handful number of miRNAs are present in both exosomal and non-exosomal portion of bovine follicular fluid. Results revealed 25 miRNAs differentially expressed (16 up and 9 down) in exosomes and 30 miRNAs differentially expressed (21 up and 9 down) in non-exosomal portion of follicular fluid in comparison of BCB- versus BCB+ oocyte groups. Expression of selected miRNAs was detected in theca, granulosa and cumulus cells that may indicate the origin of extra-cellular miRNAs in follicular fluid. To further explore the potential roles of these extra-cellular miRNAs in follicular fluid, the potential targets were predicted using in silico based analysis, and functional annotation and pathway analysis revealed most of these pathways are known regulators of follicular development and oocyte growth. In order to validate exosome mediated cell-cell communication within follicular microenvironment, we demonstrated uptake of exosomes and resulting increase of endogenous miRNA level and subsequent alteration of mRNA levels in follicular cells in vitro. The present study demonstrates for the first time, the presence of exosome or non-exosome mediated transfer of miRNA in the bovine follicular fluid, and oocyte growth dependent variation in extra-cellular miRNA signatures in the follicular environment.Exosomale und non-exosomale zirkulierende miRNAs in der Follikelflüssigkeit beim Rind: Die Rolle von exosomalen miRNAs in der Oozytenentwicklung Wachstum und Entwicklung der Rinder Follikel und Eizellen ist das Ergebnis einer Reihe von komplexen und koordinierten Prozessen, die umfangreiche Zell-Zell-Kommunikation innerhalb der Follikel beinhaltet. Diese Interaktion beruht auf komplexen und heterogenen biochemischen Stoffen sowohl in den Eizellen, den benachbarten Zellen als auch in der Follikelflüssigkeit. Die Zell-Zell-Kommunikation innerhalb der Follikel ist geprägt durch viele Signalmoleküle. Dieser Prozess kann durch Sekretion und Aufnahme von Exosomen die mehrere bioaktive Moleküle einschließlich extrazellulärer miRNAs enthalten, vermittelt werden. Bisher jedoch, sind die molekularen Mechanismen der Eizellenentwicklung und der Interaktion zwischen Eizellen und Follikelzellen in der follikulären Umgebung noch nicht voll aufgeklärt. Follikelflüssigkeit und Zellen aus einzelnen Rinder Follikeln wurden auf der Grundlage einer Vitalfärbung der entsprechenden Eizellen mittels Brillant Cresyl blau Pro gruppiert. Sowohl eine Exoquick-Präzipitation als auch differentielle Ultrazentrifugation wurde verwendet, um die exosomalen und non-exosomalen Bestandteile der Follikelflüssigkeit zu trennen. Nach miRNA Isolation von beiden Fraktionen wurde mittels des humanen miRCURY LNATMUniversal RT miRNA PCR-Array-System, ein miRNA Expressionsprofil erstellt. Durch Western-Blot-Analysen und Elektronen-Mikroskopie konnte die effiziente Trennung von exosomalen und non-exosomalen Bestandteilen in der Follikelflüssigkeit bestätigt werden. Mittels Echtzeit-qPCR-Array-Analysen konnte einige miRNAs im exosomalen und non-exosomalen Teil der Rinder Follikelflüssigkeit nachgewiesen werden. Von diesen waren 25 miRNAs im exosomalen Teil differentiell exprimiert (16 rauf- und 9 runter reguliert) und 30 miRNAs waren im non-exosomalen Teil der Follikelflüssigkeit differentiell exprimiert (21 rauf- und 9 runter reguliert) im Vergleich zu den BCB- versus BCB+ Oozyten Gruppen. Im Anschluss daran, wurde die Expression ausgewählter miRNAs in Thecazellen, Granulosa- und Kumuluszellen ermittelt. Das Ergebnis könnte ein Hinweis auf die Herkunft der extrazellulären miRNAs in der Follikelflüssigkeit sein. Für eine genauere Erklärung der möglichen Funktion dieser extrazellulären miRNAs in der Follikelflüssigkeit wurden potenziellen Zielgene mit in silico Analyse, funktioneller Annotation und Pathway-Analysen untersucht. Dies ergab, dass die miRNAs überwiegend an Signalwegen der Regulation der follikulären Entwicklung und des Eizellen Wachstums beteiligt sind. Um die Ergebnisse in Bezug auf die exosomal vermittelte Zell-Zell-Kommunikation in der follikulären Mikroumgebung zu überprüfen, konnten wir nachweisen, dass aus der Aufnahme des Exosoms in die Zelle eine Erhöhung des miRNA-Levels mit nachträglicher Veränderung des mRNA-Niveaus von Targetgenen in Follikel-Zellen in vitro resultiert. Die vorliegende Studie zeigt zum ersten Mal, den von exosomalen oder non-exosomalen Bestandteilen vermittelten Transfer von miRNA in die bovine Follikelflüssigkeit. Weiterhin ist das Wachstum der Eizellen von den Variationen der extrazellulären miRNA Signaturen in der follikulären Umgebung abhängig

Assessment of laser-assisted micromanipulation procedures in a commercial bovine in vitro production laboratory

In vitro production (IVP) of bovine embryos is increasing yearly and is rapidly becoming the most commonly-used tool in cattle breeding. The drive for more efficient food production requires accelerated dissemination of superior cattle genetics. The implementation of advanced techniques such embryo biopsy and laser assisted hatching (LAH) with IVP embryos facilitates early genetic selection and could enhance pregnancy rates following embryo transfer. The nature and extent of chromosomal errors can also be established from embryo biopsies, and this could also improve pregnancy outcomes following embryo transfer. However, the techniques of bovine embryo biopsy and LAH are laborious, time consuming, utilise expensive equipment and require a high degree of technical skill. This thesis describes a series of experiments which sought to develop easy and robust methods for embryo biopsy and LAH in a commercial laboratory setting. It assessed the survivability and ‘hatchability’ of embryos compared to those that were not manipulated

Assessment of laser-assisted micromanipulation procedures in a commercial bovine in vitro production laboratory

In vitro production (IVP) of bovine embryos is increasing yearly and is rapidly becoming the most commonly-used tool in cattle breeding. The drive for more efficient food production requires accelerated dissemination of superior cattle genetics. The implementation of advanced techniques such embryo biopsy and laser assisted hatching (LAH) with IVP embryos facilitates early genetic selection and could enhance pregnancy rates following embryo transfer. The nature and extent of chromosomal errors can also be established from embryo biopsies, and this could also improve pregnancy outcomes following embryo transfer. However, the techniques of bovine embryo biopsy and LAH are laborious, time consuming, utilise expensive equipment and require a high degree of technical skill. This thesis describes a series of experiments which sought to develop easy and robust methods for embryo biopsy and LAH in a commercial laboratory setting. It assessed the survivability and ‘hatchability’ of embryos compared to those that were not manipulated