Angus D. Macaulay

Cumulus Cell Transcripts Transit to the Bovine Oocyte in Preparation for Maturation

ABSTRACT So far, the characteristics of a good quality egg have been elusive, similar to the nature of the physiological, cellular, and molecular cues leading to its production both in vivo and in vitro. Current understanding highlights a strong and complex interdependence between the follicular cells and the gamete. Secreted factors induce cellular responses in the follicular cells, and direct exchange of small molecules from the cumulus cells to the oocyte through gap junctions controls meiotic arrest. Studying the interconnection between the cumulus cells and the oocyte, we previously demonstrated that the somatic cells also contribute transcripts to the gamete. Here, we show that these transcripts can be visualized moving down the transzonal projections (TZPs) to the oocyte, and that a time course analysis revealed progressive RNA accumulation in the TZPs, indicating that RNA transfer occurs before the initiation of meiosis resumption under a timetable fitting with the acquisition of developmental competence. A comparison of the identity of the nascent transcripts trafficking in the TZPs, with those in the oocyte increasing in abundance during maturation, and that are present on the oocytes polyribosomes, revealed transcripts common to all three fractions, suggesting the use of transferred transcripts for translation. Furthermore, the removal of potential RNA trafficking by stripping the cumulus cells caused a significant reduction in maturation rates, indicating the need for the cumulus cell RNA transfer to the oocyte. These results offer a new perspective to the determinants of oocyte quality and female fertility, as well as provide insight that may eventually be used to improve in vitro maturation conditions.

The Adenosine Salvage Pathway as an Alternative to Mitochondrial Production of ATP in Maturing Mammalian Oocytes

ABSTRACT Although the oocyte is the largest cell in the body and an unavoidable phase in life, its physiology is still poorly understood, and other cell types provide little insight into its unique nature. Even basic cellular functions in the oocyte such as energy metabolism are not yet fully understood. It is known that the mitochondria of the female gamete exhibit an immature form characterized by limited energy production from glucose and oxidative phosphorylation. We show that the bovine oocyte uses alternative means to maintain ATP production during maturation, namely, the adenosine salvage pathway. Meiosis resumption is triggered by destruction of cyclic AMP by phosphodiesterases producing adenosine monophosphate that is converted into ATP by adenylate kinases and creatine kinases. Inhibition of these enzymes decreased ATP production, and addition of their substrates restored ATP production in denuded oocytes. Addition of phosphocreatine to the oocyte maturation medium influenced the phenotype of the resulting blastocysts. We propose a model in which adenylate kinases and creatine kinases act as drivers of ATP production from added AMP during oocyte maturation.

Exploring the function of long non-coding RNA in the development of bovine early embryos

Now recognised as part of the cellular transcriptome, the function of long non-coding (lnc) RNA remains unclear. Previously, we found that some lncRNA molecules in bovine embryos are highly responsive to culture conditions. In view of a recent demonstration that lncRNA may play a role in regulating important functions, such as maintenance of pluripotency, modification of epigenetic marks and activation of transcription, we sought evidence of its involvement in embryogenesis. Among the numerous catalogued lncRNA molecules found in oocytes and early embryos of cattle, three candidates chosen for further characterisation were found unexpectedly in the cytoplasmic compartment rather than in the nucleus. Transcriptomic survey of subcellular fractions found these candidates also associated with polyribosomes and one of them spanning transzonal projections between cumulus cells and the oocyte. Knocking down this transcript in matured oocytes increased developmental rates, leading to larger blastocysts. Transcriptome and methylome analyses of these blastocysts showed concordant data for a subset of four genes, including at least one known to be important for blastocyst survival. Functional characterisation of the roles played by lncRNA in supporting early development remains elusive. Our results suggest that some lncRNAs play a role in translation control of target mRNA. This would be important for managing the maternal reserves within which is embedded the embryonic program, especially before embryonic genome activation.

Multiple Mechanisms Cooperate to Constitutively Exclude the Transcriptional Co-Activator YAP from the Nucleus During Murine Oogenesis

ABSTRACT Reproduction depends on the generation of healthy oocytes. Improving therapeutic strategies to prolong or rescue fertility depends on identifying the inter- and intracellular mechanisms that direct oocyte development under physiological conditions. Growth and proliferation of multiple cell types is regulated by the Hippo signaling pathway, whose chief effectors are the transcriptional co-activator YAP and its paralogue WWTR1. To resolve conflicting results concerning the potential role of Hippo in mammalian oocyte development, we systematically investigated the expression and localization of YAP in mouse oocytes. We report that that YAP is expressed in the germ cells beginning as early as Embryonic Day 15.5 and subsequently throughout pre- and postnatal oocyte development. However, YAP is restricted to the cytoplasm at all stages. YAP is phosphorylated at serine-112 in growing and fully grown oocytes, identifying a likely mechanistic basis for its nuclear exclusion, and becomes dephosphorylated at this site during meiotic maturation. Phosphorylation at serine-112 is regulated by a mechanism dependent on cyclic AMP and protein kinase A, which is known to be active in oocytes prior to maturation. Growing oocytes also contain a subpopulation of YAP, likely dephosphorylated, that is able enter the oocyte nucleus, but it is not retained there, implying that oocytes lack the cofactors required to retain YAP in the nucleus. Thus, although YAP is expressed throughout oocyte development, phosphorylation-dependent and -independent mechanisms cooperate to ensure that it does not accumulate in the nucleus. We conclude that nuclear YAP does not play a significant physiological role during oocyte development in mammals.

RNA Processing During Early Embryogenesis: Managing Storage, Utilisation and Destruction

The classical model of the life of a messenger RNA (mRNA) is generally depicted as a cascade of typical cellular events initiated with the transcription of the genomic sequence followed by the usual maturation of the produced transcript through splicing of the intronic regions, addition of the cap structure on its 5’ end and polyadenylation of the 3’ end. The mature mRNA is then exported out of the nucleus and sent for translation in the endoplasmic reticulum where it will serve as template/blueprint for the production of the encoded protein. The typical life cycle of an mRNA is then concluded by its decay in cellular structures that take the shape of granules called processing bodies. These well accepted steps offer a general perspective of the life and death of most mRNAs in most cellular contexts. Nonetheless, this general model does not fit well with embryogenesis mainly due to the presence of transcriptionnaly impaired cells composing the early stage embryos. In fact, the stage at which the embryo acquires the potential to transcribe its genome is widely variable between species. For instance, the mouse genome is readily activated following fertilization while in Human; transcription is initiated between the 6 and 8-cell stage. Other non-mammalian species provide more extreme situations amongst which Xenopus leavis represents a prime example of non-classical RNA management as the early embryogenesis is accomplished through 12 cell cycles conducted in the absence of transcriptional activity. In this model organism, the first embryonic cells become transcriptionally active once the embryo is composed of roughly 4,000-8,000 cells. In the absence of transcription, the embryonic cell sustains its protein production using mRNAs found in the stocks that were stored during oogenesis. These stockpiles of transcripts are accumulated during the oocyte growth that took place in the ovary, and are generally accepted as a large component of the maternal legacy that is associated with developmental competence of the resulting embryo once the egg is fertilized. The mechanisms by which the oocyte stores these transcripts are still only partly understood.