The dynamics of gene products fluctuation during bovine pre‐hatching development

Abstract

Early embryonic development, spanning fertilization to blastocyst hatching, is a very dynamic developmental window that is characterized, especially in large mammals, by a period of transcriptional incompetence that ends during the maternal to embryonic transition (MET). Prior to the MET, the first cell cycles are supported by stored RNA and proteins pools accumulated during oogenesis. Therefore, RNA and protein content are different between developmental stages. It is also known that the stability of the stored mRNA and the mechanisms for translation recruitment are partly controlled by the length of the poly(A) tail. To date, little is known about RNA and protein content fluctuations during the pre-hatching period. In this report we present measurements of total RNA, mRNA, poly(A) bearing mRNA and protein contents, as well as estimations of the proportions of both mRNA fractions to total RNA contents within these developmental stages. We found that while the ontogenic profiles of the different transcript contents were expected, their amounts were considerably lower than the reported values. Additionally, low 28S rRNA abundance and a tendency for diminishing protein content prior to the MET, suggest a limited potential for ribosomal turnover and translation. We consider the overall fluctuations in RNA and protein contents to be reference points that are essential for downstream interpretation of gene expression data across stages whether it be through candidates or high throughput approaches.

Microarray analysis of gene expression during early development: a cautionary overview

“…Following the EGA, blastomeres rapidly Pitfalls of gene expression in early embryos acquire a 'somatic' RNA management strategy in which the genome sustains basic cellular functions and also reacts and copes with the changing surrounding microenvironment. However, during the pre-EGA window, the content in the two large rRNAs is atypical of the general somatic profile (Gilbert et al 2009b). Together, these findings clearly underscore the transient state that characterizes prehatching development, during which cell size and number fluctuate in addition to transcription potential, total RNA content, proportion of each RNA type, and adenylation state of the mRNAs.…”

“…This requires the use of instruments that can handle small sample size such as the 2100 BioAnalyzer (Agilent) or the Experion System (Bio-Rad). Careful interpretation of the profiles must be done during early development because profiles observed during the early developmental stages do not fit the ones observed in the somatic cells (Gilbert et al 2009b). It is noteworthy to mention that the generally used u.v.…”

“…We have recently noticed that the well accepted in vitro transcription-based global amplification method, routinely used and implemented in high-end transcriptomic platforms, leads to a uniform yield for all samples, irrespective of their intrinsic initial RNA content. For example, it is known that in cow, the eight-cell stage has the lowest total RNA content of prehatching development, whereas the germinal stage oocyte, which contains a full stock of maternal RNAs, has a significantly higher RNA content (Lequarre et al 2004, Gilbert et al 2009b. Therefore, samples containing equal numbers of oocytes and eight-cell embryos should, if the global amplification method were truly linear, yield cDNA/aRNA levels representative of the initial differences in RNA content.…”

“…Such a situation is not possible when working with somatic cells, which thus rely on a specific amount of input RNA. Since RNA content varies widely between the stages (Gilbert et al 2009b), the physiological interpretation of across-stage results generated from samples containing equal RNA content is dependent on the knowledge of how many embryos of each developmental stage were required to produce the RNA samples. Such information is never available, making the use of samples containing a defined number of oocytes or embryos more relevant.…”

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

“…So far the functional relevance of these RNA molecules to perform specific and essential tasks in support of early embryonic development still remains unclear. As it will be discussed later, the oocyte's cytoplasm represents a rich source of messenger (Schultz and Wassarman 1977;Gilbert et al 2009) and small non-coding RNAs Review: (Krawetz 2005). Although some reports indicate a potential role of the spermatic RNA molecules, early embryonic development is undisputedly supported by the resources accumulated during oogenesis.…”