Analysis of mRNA associated factors during bovine oocyte maturation and early embryonic development

Abstract

Regulation of gene expression at the translational level is particularly essential during developmental periods, when transcription is impaired. According to the closed-loop model of translational initiation, we have analyzed components of the 5 -mRNA cap-binding complex eIF4F (eIF4E, eIF4G, eIF4A), the eIF4E repressor 4E-BP1, and 3 -mRNA poly-(A) tail-associated proteins (PABP1 and 3, PAIP1 and 2, CPEB1, Maskin) during in vitro maturation of bovine oocytes and early embryonic development up to the 16-cell stage. Furthermore, we have elucidated the activity of distinct kinases which are potentially involved in their phosphorylation. Major phosphorylation of specific target sequences of PKA, PKB, PKC, CDKs, ATM/ATR, and MAPK were observed in M II stage oocytes. Furthermore, main changes in the abundance and/or phosphorylation of distinct mRNA-binding factors occur at the transition from M II stage oocytes to 2-cell embryos. In conclusion, the results indicate that, at the transition from oocyte to embryonic development, translational initiation is regulated by striking differences in the abundance and/or phosphorylation of 5 -end and 3 -end mRNA associated factors, mainly the poly-(A) bindings proteins PABP1 and 3, their repressor PAIP2 and a Maskin-like protein with distinct eIF4E-binding properties which prevents eIF4E/cap binding and eIF4F formation in vitro. Nevertheless, from the M II stage to 16-cell embryos a substantial amount of eIF4E and, to a lesser extent, of eIF4G was precipitated by (7)m-GTP-Separose indicating eIF4F complex formation. Therefore, it is likely that in general the reduction in PABP1 and 3 abundance represses overall translation during early embryonic development.

The eIF4E repressor protein 4E‐BP2 is merely truncated, despite 4E‐BP1 degradation in the porcine uterine tissue during implantation

“…6) shows high homology in the central part of the molecule, which comprises the major phosphorylation sites at T 37/46/70 and S 65 . 4E‐BP1, however, is unique in that differential phosphorylation results in pronounced band shifts on denaturing gels (Wollenhaupt et al, 2002, 2007; Ellederova et al, 2006; Siemer et al, 2009; Shuda et al, 2011), which is not the case for 4E‐BP2 (Beretta et al, 1998; Le Bacquer et al, 2007). The two proteins cannot be distinguished by phospho‐specific antibodies due to the high sequence homology surrounding the phosphorylated sites, thus the phosphoprotein characteristics of 4E‐BP2 have not been thoroughly investigated.…”

Cytoplasmic polyadenylation in mammalian oocyte maturation

“…Maskin is a prime example of a species‐specific difference, as mammalian TACC3 lacks the eIF4E binding domain, suggesting an alternate protein or modified translational regulatory system may exist in mammals. Indeed, recent advances have revealed the potential for Maskin‐independent translational regulation . In mice, KH‐domain containing 1A ( Khdc1a ) and KH‐domain containing 1B ( Khdc1b ) were found to be expressed and translated in mouse oocytes, one of which is capable of binding eIF4e and CPEB .…”

“…In mice, KH‐domain containing 1A ( Khdc1a ) and KH‐domain containing 1B ( Khdc1b ) were found to be expressed and translated in mouse oocytes, one of which is capable of binding eIF4e and CPEB . Alternatively, another protein containing a coiled‐coil domain resembling TACC3 was discovered in cow oocytes, termed Maskin‐like, and found not to bind CPEB but able to bind eIF4e preventing cap‐binding . Although KHDC1 and Maskin‐like proteins are potential Maskin alternatives, further studies are required to elucidate their function in relation to gene‐specific regulation and whether these proteins are conserved in other mammals.…”

“…66 Alternatively, another protein containing a coiled-coil domain resembling TACC3 was discovered in cow oocytes, termed Maskin-like, and found not to bind CPEB but able to bind eIF4e preventing cap-binding. 65 Although KHDC1 and Maskin-like proteins are potential Maskin alternatives, further studies are required to elucidate their function in relation to gene-specific regulation and whether these proteins are conserved in other mammals. PAPD4 is another factor that was deemed dispensable in mouse oocytes, as knockout mice exhibited normal poly(A) tail length, suggesting there is another poly(A) polymerase that either compensates or is responsible for CP in mouse 70 despite initial studies suggesting an essential role for PAPD4.…”

The value of growth hormone supplements in ART for poor ovarian responders

“…During follicular maturation, a dialog establishes between the oocyte and the surrounding granulosa cells (GC). The growing oocyte expresses number of genes that play pivotal roles in follicular development, with oocyte presence having been recognized as indispensible for follicular development (18,19). The primary oocyte-specific factors are growth differentiation factor 9 and bone morphogenic protein 15 (20).…”