Lipid metabolism in in vitro produced embryos and epiblast-derived stem cells

Guven Ates, Gizem (2023) Lipid metabolism in in vitro produced embryos and epiblast-derived stem cells. PhD thesis, University of Nottingham.

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Infertility is a disease of the reproductive system defined as the failure to achieve a clinical pregnancy after 12 months or more of regular unprotected sexual intercourse (PierreZegers-Hochschild et al., 2009). Between 9-15% couples will have infertility problems during their lifetimes and for these infertile people, the percentage searching for medical assistance is around 56% in more developed countries and 51% in less developed countries (Boivin et al., 2007). To treat infertility, Assisted Reproductive Technologies (ARTs) were developed beginning with artificial insemination (Jouannet, 2009). Today, ART also consists of in-vitro fertilization (IVF), intracytoplasmic sperm injection and cryopreservation of germ cells (i.e., sperm, oocyte and embryos) (Sejbaek et al., 2013).

On the contrary, infertility is not common in cattle. However still ART is applied in these animals to increase food production, increase the birth of healthy offspring and store the genetic materials for future. On one hand, while high productive animals calve around eight or nine times during their lifetime, it is possible to take a few times more offspring than they will get through in their lives in a year by ART. On the other hand, ART plays a commercially important role while the import-export. Intercontinental transport of live animals cost more than transport of frozen embryos (Mapletoft and Hasler, 2005). The other fact, the ART gives us a chance to create new breeds as a productive race and expedite the genetic improvement (Wu and Zan, 2012).

The human in vitro embryo production (IVP) protocols normally commence with in vivo maturation which takes place within the ovarian follicle during gonadotropin treatment. This is because IVM is not successful enough in human ART to encourage widespread uptake (Anckaert et al., 2012). In contrast, IVM of immature oocytes is routine practice prior to fertilization in cattle IVP programs. Recently there has been a trend towards transfer of Day 5/Day 6 blastocysts instead of Day 2 cleaved zygotes in human ART (Glujovsky et al., 2016), with many cycles embracing single transfer of frozen/thawed blastocysts (Tiegs et al., 2019). This change has been developed as a result of undesirable consequences of transferred cleaved zygotes such as low implantation and birth rate and much cumulative pregnancy.

Limitations of human IVF research such as ethics and costs have led scientists to undertake animal studies. Mouse embryos have been used extensively to understand biochemical and physiology regulations of the human embryos. However, bovine and human preimplantation embryos have many similarities with respect to biochemical, paternal and maternal regulatory processes (Menezo and Herubel, 2002). Besides, bovine and human embryo development processes are completed almost at the same time. (Figure 1).

Figure 1 Comparison of embryo development stages in cattle and humans. Embryos reach the 2- and 3-cell stage around the same time. However, while human embryos form a morula around Day 4, this stage is seen on Day 5/6 in cattle. Thereafter, bovine embryo stages occur typically 1 to 2 later than human embryo stages. Credit artwork: Gizem Guven Ates

The number of cattle embryos transferred are steadily increased year on year as a consequence of improvements in bovine IVP (Figure 2A and B). The underlying reason of this development is comprehension of the metabolism of oocytes and embryos, thus determining the needs of cells viability culture. For instance, the human fluid composition of the oviduct where the zygote stays until formation of morula, demonstrates pyruvate and lactate levels significantly higher than uterus where the pre-implanted blastocyst is located. This contrasts with glucose concentrations which is over five times higher in the uterus than the oviduct and until this statement was understood required pyruvate and glucose were not placed enough in the culture system (Leese et al., 2007).

Successful production of embryo also depends on maternal and paternal factors such as age, environmental pollution and parenteral nutrition. Nowadays numerous studies have been done especially about parenteral nutrition (Sharma et al., 2020). The scientists associated parenteral nutrition with blastocyst quality, DNA methylation, metabolism of germ cells and embryos and even offspring disease risk over their lifetime. Attention was drawn to over- and undernutrition can affect sperm and seminal plasma in male (Sinclair and Watkins, 2013), metabolites in follicular environment (Robker et al., 2009), ovulation rate and embryo viability (Gonzalez-Añover et al., 2011) during preconception period.



Figure 2 Number of cattle embryos transferred by year over the past 20 years (A) (IETS, 2016) and by geographical region in 2018 (B) (IETS, 2019). IVD = in vivo derived; IVP = in vitro produced

Culture environment during in vitro maturation (IVM), fertilisation (IVF and culture (IVC) is a major limitation in the success of contemporary systems of both human and bovine IVP. An understanding of gamete/embryo metabolism is key to the successful development of these systems. There are major requirements for energy associated with biosynthetic processes and proliferation in these different cell types. Energy is provided by the breakdown of carbohydrates, proteins and lipids. Well-studied sources of energy relate to carbohydrates, such as pyruvate, glucose and lactate (Gray et al., 2014), and amino acids (Hemmings et al., 2012). The importance of lipid metabolism, however, has become increasingly appreciated in recent years (Leese, 2015). Oocytes tend to accumulate fatty acids during the terminal stages of oocyte growth prior to IVM (Sturmey et al., 2009). Fatty acid oxidation (FAO) was shown to be indispensable for oocyte meiotic maturation and developmental competence in mice (Dunning et al., 2010) although oocyte lipid content in mice is relatively low compared to that of farm animal species such as cattle and pigs (McEvoy et al., 2000). Despite differences in oocyte lipid quantity, inhibition of FAO with the carnitine palmitoyl transferase 1 (CPT1) activity inhibitor etomoxir during IVM delayed the completion of meiotic maturation in murine, bovine and porcine oocytes (Paczkowski et al., 2014). In contrast, increasing FAO by supplementation of culture media with lipid metabolism modulators such as L-carnitine, resveratrol, conjugated linoleic acid and forskolin significantly improve oocyte competence in terms of fertilization and embryo development in mice (Dunning et al., 2011) and cows (Sutton-Mcdowall et al., 2012). On the other hand, lipids are key structural components of cell membranes and form complexes with glycose and/or proteins (Muro et al., 2014). In addition, lipids are precursors of steroids which have important roles in cell signalling (Kusumi et al., 2012).

With the foregoing discussion in mind, the aim of this thesis was to improve our understanding of lipid metabolism, and fatty acid oxidation in particular, during IVM of cattle oocytes, and to investigate the long-term consequences for embryo development and viability. The ultimate goal was to generate improved systems for cattle IVP that would lead to better pregnancy outcomes following embryo transfer.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Sinclair, Kevin
Alberio, Ramiro
Keywords: Lipid metabolism, Fatty acid oxidation, Cattle oocytes, Embryo transfer, In vitro embryo production, Assisted reproductive technologies
Subjects: Q Science > QL Zoology > QL951 Embryology
Faculties/Schools: UK Campuses > Faculty of Science > School of Biosciences
Item ID: 72199
Depositing User: Guven Ates, Gizem
Date Deposited: 22 Jul 2023 04:40
Last Modified: 22 Jul 2023 04:40

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