No abstract

Microarray technologies are useful to mine the transcriptome of FCs expressed in follicles associated with competent oocytes and could be used to improve embryo selection with the objective of successful single embryo transfer.

Even after several decades of quiescent storage in the ovary, the female germ cell is capable of reinitiating transcription to build the reserves that are essential to support early embryonic development. In the current model of mammalian oogenesis, there exists bilateral communication between the gamete and the surrounding cells that is limited to paracrine signaling and direct transfer of small molecules via gap junctions existing at the end of the somatic cells' projections that are in contact with the oolemma. The purpose of this work was to explore the role of cumulus cell projections as a means of conductance of large molecules, including RNA, to the mammalian oocyte. By studying nascent RNA with confocal and transmission electron microscopy in combination with transcript detection, we show that the somatic cells surrounding the fully grown bovine oocyte contribute to the maternal reserves by actively transferring large cargo, including mRNA and long noncoding RNA. This occurrence was further demonstrated by the reconstruction of cumulus-oocyte complexes with transfected cumulus cells transferring a synthetic transcript. We propose selective transfer of transcripts occurs, the delivery of which is supported by a remarkable synapselike vesicular trafficking connection between the cumulus cells and the gamete. This unexpected exogenous contribution to the maternal stores offers a new perspective on the determinants of female fertility.

In mammals, the study of gene expression in the preimplantation embryo has been difficult because the standard procedures used to quantify mRNA generally require large amounts of starting material. The development of protocols using different quantitative strategies generally involving the polymerase chain reaction (PCR) has provided new tools for exploration of gene expression in preimplantation embryos. However, the use of an internal standard, often referred as a housekeeping gene, is essential to normalize the mRNA levels. RNA levels of eight housekeeping genes were quantified using real time PCR throughout the preimplantation period of the bovine embryo to find the most suitable gene to be used as standard. Histone H2a was the best internal standard because the transcript levels were constant across the preimplantation period. Linear amplification of antisense RNA using the T7 promotor for in vitro transcription of the entire RNA pool was evaluated as a suitable way to preamplify the starting material prior to quantification and was effective in providing accurate RNA abundance profiles throughout the preimplantation period. However, the amplification appears to be template dependent because the amplification factors were higher for some genes.

Local translation at the synapse plays key roles in neuron development and activity-dependent synaptic plasticity. mRNAs are translocated from the neuronal soma to the distant synapses as compacted ribonucleoparticles referred to as RNA granules. These contain many RNA-binding proteins, including the Fragile X Mental Retardation Protein (FMRP), the absence of which results in Fragile X Syndrome, the most common inherited form of intellectual disability and the leading genetic cause of autism. Using FMRP as a tracer, we purified a specific population of RNA granules from mouse brain homogenates. Protein composition analyses revealed a strong relationship between polyribosomes and RNA granules. However, the latter have distinct architectural and structural properties, since they are detected as close compact structures as observed by electron microscopy, and converging evidence point to the possibility that these structures emerge from stalled polyribosomes. Time-lapse video microscopy indicated that single granules merge to form cargoes that are transported from the soma to distal locations. Transcriptomic analyses showed that a subset of mRNAs involved in cytoskeleton remodelling and neural development is selectively enriched in RNA granules. One third of the putative mRNA targets described for FMRP appear to be transported in granules and FMRP is more abundant in granules than in polyribosomes. This observation supports a primary role for FMRP in granules biology. Our findings open new avenues for the study of RNA granule dysfunctions in animal models of nervous system disorders, such as Fragile X syndrome.

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 oocyte's 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.

A key step in assisted reproduction is the assessment of oocyte and embryo developmental potential in order to determine the embryo(s) most likely to result in pregnancy. Currently used embryo assessment strategies are largely based on embryo morphology and cleavage rate. Although these systems have been successful in improving pregnancy rates and reducing multiple gestations, their precision is still insufficient. Therefore, development of an objective, accurate, fast and affordable test that can aid in the assessment of oocyte and embryo developmental potential is a significant aim of reproductive medicine. Recently, global assessment strategies involving genomic, transcriptomic, proteomic or metabolomic profiling of oocytes, granulosa or cumulus cells, embryos or culture media have been applied to assisted reproduction. These technologies are at different stages of development and present unique advantages as well as limitations.

Spermiogenesis represents the transition from haploid spermatids to spermatozoa. This process entails an extreme condensation of the nucleus and a loss of nearly all cytoplasmic content. The presence of messenger RNAs in the spermatozoa has previously been shown. Generally, these transcripts are considered to be remnants of spermiogenesis. However, it has recently been proposed that there may exist a function for these sperm-associated RNAs. To address the possibility of a functional role for these transcripts, we sought to investigate and characterize the RNA pool found in bovine spermatozoa. The main goals of this study were to examine RNA integrity and survey the mRNA found in spermatids and spermatozoa. Assessment of mRNAs integrity was performed by three approaches: microelectrophoresis, comparative smearing after global amplification, and PCR amplification of target sequences located either in the 5 0 or the 3 0 ends, while mRNAs survey was performed by microarray hybridizations. RNA integrity studies in the spermatozoa showed a majority of low molecular size fragments indicating a natural segmentation of the mRNA population. The mRNA survey indicated that the sperm transcriptome harbors a complex mixture of messengers implicated in a wide array of cell functions and representing a large subset of transcripts found in spermatids. Subsequently, such sperm RNA profiling could allow the molecular diagnosis of male gamete quality.