Gene expression in mouse oocytes and preimplantation embryos: use of suppression subtractive hybridization to identify oocyte- and embryo-specific genes

The paucity of biological material has inhibited identifying genes that are differentially expressed during mammalian oogenesis and preimplantation development. We report here the linear amplification of mRNA from small numbers of mouse oocytes and preimplantation embryos to generate amounts of sense RNA that are sufficient for suppression subtractive hybridization. The resulting oocyte-specific and 8-cell-specific cDNA libraries were partially characterized, and the known oocyte-specific ZP1, ZP2, GDF-9, BMP15, and H1(oo) genes were found in the oocyte-specific cDNA library but not in the 8-cell-specific library. Further characterization of the subtracted oocyte and 8-cell embryo cDNA libraries should furnish a trove of information regarding temporal changes in gene expression during oogenesis and preimplantation development in the mouse.     

Gene expression during mammalian oogenesis and early embryogenesis: quantification of three messenger RNAs abundant in fully grown mouse oocytes

Ribonuclease protection assays have been used to quantitatively assess changes in steady-state levels of specific mRNAs during oogenesis and early embryogenesis in mice. The mRNAs encode ZP3 (a glycoprotein that serves as a sperm receptor), LDH-B (heart-type lactate dehydrogenase), and MOM-1 (a protein of unknown function). MOM-1 and LDH-B are expressed in a variety of adult mouse tissues and midgestation embryos, whereas ZP3 expression is restricted completely to oocytes. All three mRNAs are expressed by growing mouse oocytes and accumulate to unusually high levels in fully grown oocytes as compared to somatic cells; 240,000, 200,000 and 74,000 copies mRNA per fully grown oocyte for ZP3, LDH-B and MOM-1, respectively. Steady-state levels of LDH-B and MOM-1 mRNA undergo a modest decline (approximately 20-40%) during ovulation when fully grown oocytes become unfertilized eggs and, in general, mirror the reported change in poly(A)+RNA levels during this period of development. On the other hand, the level of ZP3 mRNA declines dramatically (approximately 98%) during ovulation, from approximately 240,000 copies per oocyte to approximately 5000 copies per unfertilized egg, and ZP3 mRNA is undetectable in fertilized eggs (less than 1000 copies per fertilized egg). MOM-1 mRNA is expressed at relatively low levels in morulae (approximately 2000 copies per embryo) and blastocysts (approximately 5000 copies per embryo), whereas ZP3 mRNA remains undetectable (less than 1000 copies per embryo) at these stages of preimplantation development. These findings are discussed in the context of overall gene expression during oocyte growth, meiotic maturation and early embryogenesis in mice.     

Developmental regulation and in vitro culture effects on expression of DNA repair and cell cycle checkpoint control genes in rhesus monkey oocytes and embryos

DNA repair is essential for maintaining genomic integrity, and may be required in the early embryo to correct damage inherited via the gametes, damage that arises during DNA replication, or damage that arises in response to exposure to genotoxic agents. The capacity of preimplantation stage mammalian embryos to repair damaged DNA has not been well characterized, particularly in primate embryos. In this study, we examined the expression of 48 mRNAs related to sensing different kinds of DNA damage, repairing that DNA damage, and controlling the cell cycle to provide an opportunity for DNA repair. The expression data reveal dynamic temporal changes, indicating a changing ability of the rhesus embryo to detect and repair different kinds of DNA damage. Low expression or overexpression of specific DNA repair genes may limit the ability of the embryo to respond to DNA damage at certain stages. Additionally, our data reveal that in vitro culture may lead to dysregulation of many such genes and a potentially impaired ability to repair DNA damage, thus affecting cellular viability and long-term embryo viability via effects on genome integrity. This effect of in vitro culture on nonhuman primate embryos may be relevant to assessing the potential advantages and disadvantages of prolonged in vitro culture of human embryos.     

SEBOX is essential for early embryogenesis at the two-cell stage in the mouse

Previously, we found high levels of skin-embryo-brain-oocyte homeobox (Sebox) gene expression in germinal vesicle (GV)-stage oocytes. The objective of the present study was to determine the role played by SEBOX in oocyte maturation and early embryogenesis using RNA interference (RNAi). Microinjection of Sebox double-stranded RNA into GV oocytes resulted in a marked decrease in Sebox mRNA and protein expression. However, Sebox RNAi affects neither oocyte maturation rate nor morphological characteristics, including spindle and chromosomal organization of metaphase II oocytes. In addition, Sebox RNAi had no discernible effect on the activities of M-phase promoting factor or mitogen-activated protein kinase. In contrast, microinjection of Sebox double-stranded RNA into pronuclear-stage embryos resulted in holding embryo development at the two-cell (84.9%) and the four- and eight-cell (15.1%) stages. We concluded that Sebox is a new addition to maternal effect genes that produced and stored in oocytes and function in preimplantation embryo development.     

A potential role for triglyceride as an energy source during bovine oocyte maturation and early embryo development

The potential role of endogenous triglyceride in bovine oocyte maturation and preimplantation development has been investigated. Bovine immature oocytes were recovered from abattoir-derived ovaries, matured and fertilised in vitro and the zygotes grown to the blastocyst stage in SOFaaBSA. Methyl palmoxirate (MP) blocks the oxidation of fatty acids by inhibiting mitochondrial carnitine palmitoyltransferase A. The development of zygotes exposed to MP during oocyte maturation, and of zygotes exposed to MP during embryo culture has been assessed in terms of oxygen consumption by oocytes and embryos during a 4-6 hr incubation period in the presence of MP and as blastocyst formation and cell number. Immature oocytes exposed to MP during maturation had reduced capacity to form blastocysts after fertilisation; the same effect was apparent, but to a lesser extent, in zygotes exposed to MP during embryo development. Oxygen consumption values of oocytes and blastocysts in the absence of exogenous substrates were similar to those in control medium containing nutrients. MP-inhibited oxygen consumption of immature oocytes, mature oocytes, cleavage stages embryos and blastocysts by 64, 45, 12 and 13%, respectively. The data are consistent with a role for triglyceride as a key energy source during bovine oocyte maturation and potentially, during preimplantation embryo development.     

A genome-wide study of gene activity reveals developmental signaling pathways in the preimplantation mouse embryo

The preimplantation development of the mammalian embryo encompasses a series of critical events: the transition from oocyte to embryo, the first cell divisions, the establishment of cellular contacts, the first lineage differentiation-all the first subtle steps toward a future body plan. Here, we use microarrays to explore gene activity during preimplantation development. We reveal robust and dynamic patterns of stage-specific gene activity that fall into two major phases, one up to the 2-cell stage (oocyte-to-embryo transition) and one after the 4-cell stage (cellular differentiation). The mouse oocyte and early embryo express components of multiple signaling pathways including those downstream of Wnt, BMP, and Notch, indicating that conserved regulators of cell fate and pattern formation are likely to function at the earliest embryonic stages. Overall, these data provide a detailed temporal profile of gene expression that reveals the richness of signaling processes in early mammalian development.     

Vitrification of murine mature metaphase II oocytes perturbs DNA methylation reprogramming during preimplantation embryo development

Accurate reprogramming of DNA methylation occurring in preimplantation embryos is critical for normal development of both fetus and placenta. Environmental stresses imposed on oocytes usually cause the abnormal DNA methylation reprogramming of early embryos. However, whether oocyte vitrification alters the reprogramming of DNA methylation (5 mC) and its derivatives in mouse preimplantation embryo development remains largely unknown. Here, we found that the rate of cleavage and blastocyst formation of embryos produced by IVF of vitrified matured oocytes was significantly lower than that in control counterparts, but the quality of blastocysts was not impaired by oocyte vitrification. Additionally, although vitrification neither altered the dynamic changes of 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5 fC) before 4-cell stage nor affected the levels of 5 mC and 5-carboxylcytosine (5caC) throughout the preimplantation development, vitrification significantly reduced the levels of 5hmC and 5 fC from 8-cell stage onwards. Correspondingly, vitrification did not alter the expression patterns of Tet3 in preimplantation embryos but apparently reduced the expression levels of Tet1 in 4-cell and 8-cell embryos and increased the expression levels of Tet2 at morula stage. Taken together, these results demonstrate that oocyte vitrification perturbs DNA methylation reprogramming in mouse preimplantation embryo development.