TAF4b, a TBP associated factor, is required for oocyte development and function

Development of a fertilizable oocyte is a complex process that relies on the precise temporal and spatial expression of specific genes in germ cells and in surrounding somatic cells. Since female mice null for Taf4b, a TBP associated factor, are sterile, we sought to determine when during follicular development this phenotype was first observed. At postnatal day 3, ovaries of Taf4b null females contained fewer (P < 0.01) oocytes than ovaries of wild type and heterozygous Taf4b mice. However, expression of only one somatic cell marker Foxl2 was reduced in ovaries at day 15. Despite the reduced number of follicles, many proceed to the antral stage, multiple genes associated with granulosa cell differentiation and oocyte maturation were expressed in a normal pattern, and immature Taf4b null females could be hormonally primed to ovulate and mate. However, the ovulated cumulus oocyte complexes from the Taf4b null mice had fewer (P < 0.01) cumulus cells, and the oocytes were functionally abnormal. GVBD and polar body extrusion were reduced significantly (P < 0.01). The few oocytes that were fertilized failed to progress beyond the two-cell stage of development. Thus, infertility in Taf4b null female mice is associated with defects in early follicle formation, oocyte maturation, and zygotic cleavage following ovulation and fertilization.     

Somatic cell-like features of cloned mouse embryos prepared with cultured myoblast nuclei

Cloning by somatic cell nuclear transfer requires silencing of the donor cell gene expression program and the initiation of the embryonic gene expression program (nuclear reprogramming). Failure to silence the donor cell program could lead to altered embryonic phenotypes. Cloned mouse embryos produced using myoblast nuclei fail to thrive in standard embryo culture media but flourish in somatic cell culture media favored by the donor myoblasts themselves, forming blastocysts at a significant rate, with robust morphologies, high total cell number, and a normal allocation of cells to the inner cell mass in most embryos. Myoblast cloned embryos continue expressing the GLUT4 glucose transporter, which is typically expressed in muscle but not in preimplantation stage embryos. Myoblast clones also exhibit precocious enrichment of GLUT1 at the cell surface. Both myoblast and cumulus cell cloned embryos exhibit enhanced rates of glucose uptake. These observations indicate that silencing of the donor cell genome during cloning either is incomplete or occurs progressively over the course of preimplantation development. As a result, cloned embryos initially exhibit many somatic cell-like characteristics. Tetraploid constructs, which possess a transplanted somatic cell genome plus the oocyte-derived chromosomes, exhibit a more embryonic-like pattern of gene expression and culture preference. We conclude that preimplantation stage cloned embryos have profoundly altered characteristics that are donor cell type specific and that exposure of cloned embryos to standard embryo culture conditions may lead to disruptions in basic homeostasis and inhibition of a range of essential processes including further nuclear reprogramming, contributing to cloned embryo demise.     

Incomplete reactivation of Oct4-related genes in mouse embryos cloned from somatic nuclei

The majority of cloned animals derived by nuclear transfer from somatic cell nuclei develop to the blastocyst stage but die after implantation. Mouse embryos that lack an Oct4 gene, which plays an essential role in control of developmental pluripotency, develop to the blastocyst stage and also die after implantation, because they lack pluripotent embryonic cells. Based on this similarity, we posited that cloned embryos derived from differentiated cell nuclei fail to establish a population of truly pluripotent embryonic cells because of faulty reactivation of key embryonic genes such as Oct4. To explore this hypothesis, we used an in silico approach to identify a set of Oct4-related genes whose developmental expression pattern is similar to that of Oct4. When expression of Oct4 and 10 Oct4-related genes was analyzed in individual cumulus cell-derived cloned blastocysts, only 62% correctly expressed all tested genes. In contrast to this incomplete reactivation of Oct4-related genes in somatic clones, ES cell-derived cloned blastocysts and normal control embryos expressed these genes normally. Notably, the contrast between expression patterns of the Oct4-related genes correlated with efficiency of embryonic development of somatic and ES cell-derived cloned blastocysts to term. These observations suggest that failure to reactivate the full spectrum of these Oct4-related genes may contribute to embryonic lethality in somatic-cell clones.     

小鼠母源因子对早期胚胎发育的影响

在脊椎动物中发育过程中,卵母细胞要经历MII期停滞、受精、早期胚胎发育的启动、胚胎基因组的转录激活、并指导完成个体的发育过程。同时,核移植过程中,分化的细胞核在去核的卵母细胞中能够重编程到胚胎早期的状态并能完成个体的发育过程。在这些发育过程中母源因子都发挥了极其的重要作用。在小鼠胚胎发育研究中发现,小鼠的基因组激活发生在2细胞期,这一时期标志着合子的发育由卵母细胞控制向胚胎控制的过渡,期间发生一系列复杂的生化过程。体外培养的小鼠的胚胎的发育阻断也易发生的2细胞时期。因此对卵母细胞及早期胚胎母源因子的研究,将有利于了解早期体外培养胚胎和克隆胚胎发育失败的原因,为提高体外培养和克隆胚胎发育的成功率提供理论的基础。     

Expression of beta-galactosidase in preimplantation ovine and porcine embryos

Knowledge regarding the timing of embryonic expression of the mammalian genome is of relevance for the development of preimplantation diagnostic methods for human genetic diseases. For development of preimplantation diagnosis of lysosomal storage diseases, it will be necessary to know at which embryonic stage the genes for lysosomal enzymes are expressed. In previous studies by other investigators, it has been shown that lysosomal alpha- and beta-galactosidase and beta-glucuronidase in murine embryos increase 50- to 100-fold in activity between the two-cell and late blastocyst stage. We describe here expression of lysosomal beta-galactosidase in preimplantation ovine (two-cell through midblastocyst) and porcine (two-cell through late blastocyst) embryos. Expression of beta-galactosidase in ovine and porcine preimplantation embryos followed a similar rate of increase as that described for murine embryos. Activity of beta-galactosidase increased over 10-fold between the two- to four-cell and midblastocyst stages in ovine embryos, and 300-fold between the two- to four-cell and late blastocyst stages in porcine embryos. Activity expressed on a per cell basis was relatively constant in ovine embryos, as has been described in murine embryos, and increased approximately 5-fold on a per cell basis in porcine embryos. Activity of beta-galactosidase in ovine and porcine embryos initially was greater than 12-fold on a per cell or per embryo basis than in murine embryos evaluated. The knowledge of beta-galactosidase embryonic expression may provide the basis for preimplantation diagnosis of genetic beta-galactosidase deficiency in these species.     

Expression and regulation of genes associated with cell death during murine preimplantation embryo development

The newly fertilized preimplantation embryo depends entirely on maternal mRNAs and proteins deposited and stored in the oocyte prior to its ovulation. If the oocyte is not sufficiently equipped with maternally stored products, or if zygotic gene expression does not commence at the correct time, the embryo will die. One of the major abnormalities observed during early development is cellular fragmentation. We showed previously that cellular fragmentation in human embryos can be attributed to programmed cell death (PCD). Here, we demonstrate that the PCD that occurs during the 1-cell stage of mouse embryogenesis is likely to be regulated by many cell death genes either maternally inherited or transcribed from the embryonic genome. We have demonstrated for the first time the temporal expression patterns of nine cell death regulatory genes, and our preliminary experiments show that the expression of these genes is altered in embryos undergoing fragmentation. The expression of genes involved in cell death (MA-3, p53, Bad, and Bcl-xS) seems to be elevated, whereas the expression of genes involved in cell survival (Bcl-2) is reduced. We propose that PCD may occur by default in embryos that fail to execute essential developmental events during the first cell cycle. Mol. Reprod. Dev. 51:243–253, 1998. © 1998 Wiley-Liss, Inc.