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Function, Expression and Glucose-dependent Regulation of Monocarboxylate-Proton Co-transporter molecules (MCT) in Mouse Preimplantation Development.

ABSTRACT The purpose of this project was to investigate monocarboxylate (i.e. pyruvate and lactate) transport in the preimplantation stage of embryo development. Much progress has been made over the last 15 years towards understanding preimplantation and peri-implantation embryo physiology, including metabolic preferences during this period. It is known that as the cells (blastomeres) of an embryo compact via tight junctions and the embryo differentiates into a blastocyst, a metabolic “switch” occurs to allow the blastocyst to take up glucose at a rapid rate, obtaining energy derived from glycolysis. Glucose transporter molecules have been identified and characterized during this period of development and a paradigm for glucose transport has been described. However, during the early cleavage stages (days 1-3 post-fertilization), the embryo preferentially derives its metabolic energy from the monocarboxylate pyruvate. Evidence for the expression of pyruvate transporter molecules (a family of proton-coupled monocarboxylate co-transporters, MCT) has only been indicated via some kinetic studies on pH homeostasis and PCR analysis for MCT expression, and results have been conflicting (Gibb et al., 1997, Harding et al., 1999, Herubel et al., 2002). This project aimed to clarify discrepancies in reports for mRNA expression of MCT and to enhance the understanding of monocarboxylate transport processes during preimplantation development by pioneering investigations into protein expression for various MCT isoforms. Transport kinetics for monocarboxylate, DL-lactate, were examined by measuring the uptake of radioactive [3H]-DL-lactate from the medium by two-cell embryos and blastocysts. It was discovered that blastocysts demonstrate significantly higher affinity for DL-lactate compared to zygotes (Km 20 + 10 v 87 + 35 mM lactate; p=0.03), which suggested that alterations in the expression of various MCT isoforms might be expected as the embryo developed to a blastocyst. The rate of transport showed a trend towards a decrease from the zygote to blastocyst stages, although this could not be confirmed as significant within the limitations of this experiment. Mouse embryos, both in vivo and in vitro-derived, were collected and pooled at the zygote, two-cell, morula and blastocyst stages of development. RNA purification, reverse-transcription and PCR were used to analyze the expression of the four best-characterized MCT isoforms. MCT1, MCT2 and MCT4 were all found to be expressed in oocytes and mouse embryos from the zygote through to the preimplantation blastocyst. MCT3, an isoform uniquely expressed in the retina, was not detected at any stage in embryos. Since glucose has been implicated in regulatory processes involving glucose transporter expression in mouse embryos (Pantaleon et al., 2005, Pantaleon et al., 2001), mRNA expression was examined in the presence or absence of glucose in the culture media to determine whether the same phenomena applied to MCT. It was discovered that MCT1 and MCT4 isoforms were responsive to glucose-deprivation as evidenced by a reduction in mRNA expression in compacted morula cultured from the zygote stage without glucose. When glucose-deprived embryos were exposed to a brief high concentration of glucose during the 4-cell stage of development and continued in culture without glucose, the expression of mRNA for MCT1 and MCT4 persisted post-compaction, demonstrating that glucose exposure is necessary for the continued expression of these two isoforms in the mouse blastocyst. MCT2 mRNA did not respond to the absence of glucose in this way, and mRNA expression persisted in either the presence or absence of glucose. To follow these analyses of MCT gene transcription during early embryo development, confocal laser scanning immunofluorescence and western blotting were used to identify the expression of MCT proteins at various stages of development. Culture in the presence or absence of glucose was again employed to determine whether the changes seen in mRNA expression were conveyed at the protein level. All three proteins were identified throughout preimplantation development, though their locations were uniquely different. MCT1 was notably absent from plasma membranes at all stages, and was detected diffusely within the cytoplasm. In expanding blastocysts MCT1 tended to concentrate in the cortical cytoplasm of blastomeres and staining was more intense in the polar trophectoderm. In this cytoplasmic location its function is unclear. MCT1 does not appear to be a key transporter of monocarboxylates into and out of the embryo, but it may have a role in shuttling pyruvate and lactate within the cytoplasm to maintain metabolic and redox homeostasis. In embryos cultured without glucose, the immunostaining intensity for MCT1 gradually decreased as morulae degenerated and died. Protein loss occurred from the morula stage onwards, whilst mRNA was already undetectable at this stage. This would indicate that glucose signals which maintain mRNA expression most likely operate at the level of gene activation/transcription with latent effects on protein expression. MCT4 appeared to be located on the plasma membranes of oocytes and 2-cell embryos and nuclear staining was evident throughout preimplantation development, however plasma membrane expression was not apparent in morulae and blastocysts. This is consistent with earlier kinetic evidence of a low affinity lactate transporter (Km 87 + 35 mM lactate) operating at the early preimplantation stages. MCT4 has the lowest affinity for lactate of all the characterized MCT to date. Kinetic data also suggests that a change might occur in MCT protein expression as the embryo progresses to a blastocyst with a higher affinity lactate transporter taking precedence, and the loss of MCT4 from the plasma membrane at these later stages supports this view. Similarly to MCT1, MCT4 mRNA expression was also found to be dependent on glucose exposure during the early preimplantation period, and embryos cultured entirely without glucose demonstrated a loss of MCT4 mRNA expression at the morula stage. MCT4 typically exists as a lactate exporter in glycolytic tissues and it most likely exports lactate from the embryo for pH and redox homeostasis during this period of development. Protein localization studies found MCT2 to be located on the plasma membranes of oocytes, zygotes, 2-cell embryos, and polarized to the surface of the outer blastomeres of morulae and blastocyst trophectodermal cells. Throughout preimplantation development, MCT2 protein co-localized with peroxisomal catalase in peroxisome-sized granules throughout the cells. Known to be a high affinity pyruvate transporter, given its location in embryos it was proposed here that MCT2 most likely imports pyruvate to fuel early embryos, and later works as a bifunctional pyruvate/lactate importer/exporter on the transporting epithelium (trophectoderm) of blastocysts to maintain the pH, redox and metabolic status of the embryo. MCT2 was an enigma to the other MCT. Its expression in the absence of glucose behaved in an opposite way to that of MCT1 and MCT4, with mRNA expression persisting in the absence of glucose. In fact, MCT2 and catalase proteins demonstrated a quantitative increase in embryos lacking glucose, and the increase in staining was noticed as an increase in the density of peroxisome-like structures (or peroxisome proliferation) within the embryo. As such, it was decided to investigate the possibility that peroxisome proliferators (Peroxisome Proliferator Activated Receptors, PPARs) were involved in the control of MCT expression in the same way that they are known to control the expression of catalase and other peroxisomal proteins. At this stage, no MCT isoforms had been identified as being under the control of PPARs, although it was known that their expression was most likely controlled at the level of transcription, with no translational or post-translational controlling elements. PPARα, one of three isoforms (α, γ and β/δ) was selected as a likely candidate given that it controls peroxisomal proliferation and fatty acid β-oxidation processes at the level of transcription in other tissues, and it was known to be upregulated in conditions of starvation and oxidative stress. PPARα mRNA was shown to be expressed in early cleavage preimplantation mouse embryos, but its expression was reduced in morulae and blastocysts. Further, lack of glucose led to persistence of PPARα mRNA expression at the morula stage. PPARα protein was also demonstrated to stain more brightly in early preimplantation embryos compared to later stages. Further experimentation demonstrated that the phenomenon of increased catalase and MCT2 expression in embryos cultured without glucose could be mimicked in the presence of glucose by treating these embryos with the PPARα-selective agonist, WY14,643. The timing and quantitative nature of this upregulation were very similar, suggesting that PPARα was in some way involved in the glucose-deprived upregulation pathway for catalase and MCT2. To further investigate this pathway, oxidative stress was investigated in embryos cultured in the presence and absence of glucose to test whether the generation of reactive oxygen species contributed to the PPARα/MCT2 phenomenon. It was demonstrated that within 2 h of culture in the absence of glucose, hydrogen peroxide levels were significantly elevated in zygotes. Amelioration of increased peroxide generation in glucose-deprived embryos using a non-selective flavoenzyme inhibitor diphenyleneiodonium (DPI) eliminated any increases in PPARα and MCT2 protein expression that were earlier noted in the absence of glucose. To summarize, MCT1, MCT2 and MCT4 mRNA and protein expression were successfully demonstrated in mouse preimplantation embryos and all were confirmed to be in some way regulated by glucose in the culture medium. In the absence of glucose, mRNA expression for MCT1 and MCT4 were reduced to undetectable levels in morulae indicating that their expression was glucose-dependent. Paradoxically, glucose deprivation caused an increase in PPARα, catalase and MCT2 protein expression. PPARα-selective agonism in the presence of glucose induced similar timing and effects on catalase and MCT2 upregulation, implicating PPARα in this pathway. Hydrogen peroxide levels were significantly elevated within 2 h of culture in the absence of glucose. This peroxide elevation could be quenched to control levels by treating these embryos with DPI, and reducing hydrogen peroxide to control levels also eliminated the upregulation of PPARα and MCT2, implicating oxidative stress as an important component in the glucose-deprivation induced upregulation of MCT2. The experimental data presented in this thesis demonstrate that from its very conception, the embryo interacts with, adapts to, and is indeed affected by the external environment in which it develops. Even components like glucose, once considered simply as metabolic substrates, have profound effects on gene transcription and protein expression within the embryo which may impact on later its developmental competence, a reality we need to consider more deeply in light of the implementation of artificial reproductive technologies widely used today in zoology, agriculture and clinically, in humans.