During early mammalian development, genesis of the first two cell lineages, inner cell mass (ICM) and trophectoderm (TE), is dependent upon functions of key transcription factors that are expressed in a regulated and spatially restricted fashion. In this study, we demonstrate that during early mouse development, mRNA expression of transcription factor GATA3 is induced at the 4-cell stage and is consistently present during pre-implantation embryonic development. Interestingly, at the blastocyst stage, Gata3 mRNA is selectively up-regulated within the TE lineage, and GATA3 protein is abundantly present only in the TE but not in the ICM. Using mouse trophoblast stem cells (TS cells) as a model, we found that, knockdown of GATA3 by RNA interference (RNAi) down-regulates expression of caudal-type homeobox 2 (CDX2), a key regulator of the TE lineage. Chromatin immunoprecipitation (ChIP) analyses revealed that, in TS cells, GATA3 directly regulates Cdx2 transcription from a conserved GATA motif at the intron 1 region of the Cdx2 locus. ChIP analyses with mouse blastocysts also detected GATA3 occupancy at intron 1 of the Cdx2 locus. In addition, downregulation of GATA3 in pre-implantation mouse embryos reduces Cdx2 expression and inhibits morula to blastocyst transformation. Our results indicate a novel function of GATA3, in which it is selectively expressed in TE, regulates expression of key genes in TE lineage, and is involved in morula to blastocyst transformation. Genesis of the trophectoderm (TE)2 and inner cell mass (ICM) lineages during early mouse development appears to occur in two stages (1-3). First, cells are allocated to different inside and outside positions via asymmetric divisions. Then, the cells in these different positions become specified, and they become committed to restricted developmental fates. Outside cells become committed to the TE, and inside cells become ICM. Development of ICM and TE is regulated by key transcription factors that specify TE and ICM cell fate, and CDX2 has been implicated in this process (4 -6). Multiple studies indicated the importance of CDX2 in TS cell proliferation, proper function of TE, and successful implantation of blastocyst (5-8). However, molecular mechanisms that regulate Cdx2 expression in trophoblast cell lineages are poorly understood. Two other transcription factors, eomesodermin (Eomes) and TEA domain family member 4 (TEAD4), are also implicated in TE development. Mutation studies showed that the lack of Eomes also arrests blastocyst development (9). However, Cdx2 is still expressed in Eomes mutants (5). Tead4 mutants show more severe phenotypes than Cdx2 mutants and are characterized by loss of Cdx2 expression (10, 11). However, unlike Cdx2, Tead4 expression is not restricted to the TE lineage during pre-implantation development indicating that additional regulatory mechanisms are involved for the restricted expression of Cdx2 in TE lineage.Earlier, we found that, among the six members (GATA1-6) of GATA family of transcription factors, only GATA3 is abunda...

In the preimplantation mouse embryo, TEAD4 is critical to establishing the trophectoderm (TE)-specific transcriptional program and segregating TE from the inner cell mass (ICM). However, TEAD4 is expressed in the TE and the ICM. Thus, differential function of TEAD4 rather than expression itself regulates specification of the first two cell lineages. We used ChIP sequencing to define genomewide TEAD4 target genes and asked how transcription of TEAD4 target genes is specifically maintained in the TE. Our analyses revealed an evolutionarily conserved mechanism, in which lack of nuclear localization of TEAD4 impairs the TE-specific transcriptional program in inner blastomeres, thereby allowing their maturation toward the ICM lineage. Restoration of TEAD4 nuclear localization maintains the TE-specific transcriptional program in the inner blastomeres and prevents segregation of the TE and ICM lineages and blastocyst formation. We propose that altered subcellular localization of TEAD4 in blastomeres dictates first mammalian cell fate specification.A llocation of blastomeres to outside and inside positions during preimplantation mammalian development initiates specification of the first two cell lineages, the trophectoderm (TE) and the inner cell mass (ICM) (1, 2). Successful progression of TE and ICM fate specification and proper development of the preimplantation embryo depends on differential transcriptional programs that are instigated and maintained within the outer and inner cells. Gene-KO studies in mice showed TEAD4 as the master orchestrator of the TE-specific transcriptional program (3-5). TEAD4-null embryos do not mature to the blastocyst stage and TEAD4-null blastomeres lack expression of TE-specific master regulators like CDX2, GATA3, and EOMES (3, 4). However, they maintain expression of ICM-specific factors like OCT4 and NANOG.Interestingly, TEAD4 expression is maintained both in cells of TE and ICM lineages, as well as in the TE-derived trophoblast stem cells (TSCs) and ICM-derived ES cells (ESCs) (5, 6). Thus, questions are raised as to how TEAD4 selectively orchestrates the TE/TSC-specific transcriptional program but not the ICM/ ESC-specific transcriptional program. The current model predicts that the presence vs. the absence of a TEAD4 cofactor, yesassociated protein (YAP), modulates TEAD4 function at its target genes in outer vs. inner blastomeres (6), leading to the segregation of the TE and ICM lineages. However, YAP-null mouse embryos do not show preimplantation developmental defects (7), indicating that, unlike TEAD4, YAP function is dispensable during TE and ICM fate determination. It is proposed that another YAP-related cofactor, WWTR1 (i.e., TAZ), could compensate for the absence of YAP during early development (6). However, the mode of TAZ function during TE and ICM specification is unknown. Furthermore, direct targets of TEAD4 have not been identified in the TE or in trophoblast cells. Thus, definitive experiments have not been performed to conclude that loss of cofactor function/recruitmen...

IFN-is a secretory product of trophectoderm of cattle, sheep, and their relatives and is expressed for a few days in early pregnancy after the blastocyst first forms. It serves to alert the mother that she is pregnant. A delayed or less than robust IFN-signal is a likely cause of embryonic loss. Here we have determined whether blastocyst production of IFN-, which is encoded by a cluster of genes on chromosome 9, differs between the sexes in cattle, as assessed by culture of in vitro-derived embryos on two different media, one complex (tissue culture medium 199 supplemented with serum) with coculture support, the other relatively simple (synthetic oviductal fluid plus albumin). With both media, female blastocysts produced approximately double the amount of IFN-as males, regardless of such variables as oocyte batch, blastocyst quality, hatching, and length of time in culture. However, in either tissue culture medium 199, which contains 5.5 mM D-glucose, or in synthetic oviductal fluid, in the presence but not in the absence of added glucose, significantly fewer female than male embryos were able to progress from the morula͞early blastocyst stage to more advanced stages of development. It is possible that the differences between male and female embryos both in their production of IFN-and in their ability to progress in development in glucose-rich media are manifestations of phenomena that occur in vivo and provide plasticity in embryo selection during early pregnancy.embryo culture ͉ in vitro maturation-in vitro fertilization ͉ sexual dimorphism

The intricate molecular mechanisms that regulate embryonic stem (ES) cell pluripotency are incompletely understood. Prior research indicated that activation of JAK-STAT3 pathway or inhibition of ERK/GSK3 signaling maintains mouse ES cell (mESC) pluripotency. Here we demonstrate that inhibition of protein kinase C (PKC) isoforms maintains mESC pluripotency without the activation of STAT3 or inhibition of ERK/GSK3 signaling pathways. Our analyses revealed that the atypical PKC isoform, PKCζ plays an important role in inducing lineage commitment in mESCs through a PKCζ–NF-κB signaling axis and inhibition of PKC isoforms maintains ES cell-specific epigenetic modifications. Furthermore, inhibition of PKC isoforms permits derivation of germline-competent ES cells from mouse blastocysts and also facilitates reprogramming of mouse embryonic fibroblasts (MEFs) towards induced pluripotent stem cells (iPSCs). Our results indicate that PKC signaling is critical to balancing ES cell self-renewal and lineage commitment.

The human Zip4 gene (Slc39a4) is mutated in the rare recessive genetic disorder of zinc metabolism acrodermatitis enteropathica, but the physiological functions of Zip4 are not well understood. Herein we demonstrate that homozygous Zip4-knockout mouse embryos die during early morphogenesis and heterozygous offspring are significantly underrepresented. At mid-gestation, an array of developmental defects including exencephalia, anophthalmia and severe growth retardation were noted in heterozygous embryos, and at weaning, many (63/280) heterozygous offspring were hydrocephalic, growth retarded and missing one or both eyes. Maternal dietary zinc deficiency during pregnancy exacerbated these effects, whereas zinc excess ameliorated these effects and protected embryonic development of heterozygotes but failed to rescue homozygous embryos. Heterozygous Zip4 embryos were not underrepresented in litters from wild-type mothers, but were approximately 10 times more likely to develop abnormally than were their wild-type littermates during zinc deficiency. Thus, both embryonic and maternal Zip4 gene expressions are critical for proper zinc homeostasis. These studies suggest that heterozygous mutations in the acrodermatitis gene Zip4 may be associated with a wider range of developmental defects than was previously appreciated, particularly when dietary zinc is limiting.

This study was designed to examine the relationship between the speed at which bovine embryos reach the blastocyst stage, their cell number, and interferon-tau production. A total of 800 oocytes were fertilized by frozen-thawed semen. On day 2, 44 hr after exposure to sperm, 78, 320, and 296 embryos were at the two-, four-, and eight-cell stages, respectively, with an overall cleavage rate of 86.8%. Within these three groups 15 (19.2%), 106 (33.1%), and 158 (53.4%) embryos proceeded to the blastocyst stage. Of these 46.7%, 65.1%, and 63.3% hatched in the three groups, respectively. Blastocysts began to appear at day 7, but a few did not form until as late as day 13. Expanded blastocysts (n = 279) were cultured individually for 48 hr in 50-microliter droplets of medium, fixed for cell counts, and the concentration of interferon-tau in the medium was determined. Blastocysts originating from two-cell embryos had significantly fewer cells (46.5 +/- 23.3) than either four-cell- (97.2 +/- 13.5) or eight-cell-derived embryos (113.8 +/- 13.6; P < 0.05). Hatching was accompanied by an increase in cell number (129.8 +/- 15.5 versus 41.9 +/- 14.4; P < 0.01). Blastocysts derived from embryos that had reached the eight- or four-cell stage 44 hr after insemination produced significantly more interferon than embryos derived from two-cell embryos (941.7 +/- 92.1, 930.1 +/- 163.1, versus 232.8 +/- 70.1 pM). In contrast, hatching, ovary batch, the speed of early cleavage, cell number, and quality grade had no effect on interferon-tau secretion. The embryo's age at blastocyst formation was not related to the number of its cells but did have a significant effect (P < 0.001) on interferon-tau production, with mean concentrations in the medium of 294.8 +/- 57.9, 563.3 +/- 82.0, 1126.3 +/- 133.6, 1778.5 +/- 297.2, 512.9 +/- 82.0, 315.0 +/- 157.5, and 157.5 pM among blastocysts appearing from days 7 to 13, respectively. These data suggest that blastocysts that form at days 7 and 8 produce less interferon-tau than those that form on days 9 or 10. Since early-forming blastocysts are generally considered more developmentally competent than those which form late, there may be a negative relationship between early interferon-tau production and competence.

FSH and luteinizing hormone (LH) are secreted constitutively or in pulses, respectively, from pituitary gonadotropes in many vertebrates, and regulate ovarian function. The molecular basis for this evolutionarily conserved gonadotropin-specific secretion pattern is not understood. Here, we show that the carboxyterminal heptapeptide in LH is a gonadotropin-sorting determinant in vivo that directs pulsatile secretion. FSH containing this heptapeptide enters the regulated pathway in gonadotropes of transgenic mice, and is released in response to gonadotropin-releasing hormone, similar to LH. FSH released from the LH secretory pathway rescued ovarian defects in Fshb-null mice as efficiently as constitutively secreted FSH. Interestingly, the rerouted FSH enhanced ovarian follicle survival, caused a dramatic increase in number of ovulations, and prolonged female reproductive lifespan. Furthermore, the rerouted FSH vastly improved the in vivo fertilization competency of eggs, their subsequent development in vitro and when transplanted, the ability to produce offspring. Our study demonstrates the feasibility to fine-tune the target tissue responses by modifying the intracellular trafficking and secretory fate of a pituitary trophic hormone. The approach to interconvert the secretory fate of proteins in vivo has pathophysiological significance, and could explain the etiology of several hormone hyperstimulation and resistance syndromes.protein sorting | regulated secretion | dense core granules | folliculogenesis

The effects of culturing bovine embryos in groups were investigated. In the first experiment, 1000 oocytes were matured, fertilized and then cultured in groups of 40 in 25 microl of medium. From half of these groups, blastocysts were removed and cultured separately, while in the other half blastocysts were allowed to remain in the group culture microdrop. Blastocysts developed equally well in both groups, although hatching was reduced in those blastocysts removed from the culture droplet. In the second experiment, 1000 zygotes were cultured from the 8-cell stage to the blastocyst stage either individually or in groups of 40. Culture in groups increased the formation of blastocysts, the percentage of hatching blastocysts, the number of cells within blastocysts and the production of interferon-tau. In the final experiment, 1000 zygotes were cultured in groups up to the blastocyst stage. Two-thirds of these blastocysts were then cultured in groups of three, while the remaining blastocysts were cultured individually. Co-culture did not affect hatching or cell number but significantly elevated interferon-tau secretion. These results demonstrate that group culture either before or after blastocyst formation can alter the expression of a specific gene important for the establishment of pregnancy.