Lineage-specific expression of heterochromatin protein 1gamma in post-compaction, in vitro-produced bovine embryos.

Heterochromatin protein 1gamma (HP1gamma) is a highly conserved regulator of euchromatic and heterochromatic gene expression. Mammalian HP1gamma is essential for both successful preimplantation embryo development and maintenance of pluripotency in embryonic stem cells in vitro. Here, we describe HP1gamma protein localisation in matured (MII) bovine oocytes and IVF preimplantation embryos at defined developmental stages. HP1gamma is expressed in post-compaction embryos in a highly lineage-specific pattern. In embryonic stages preceding the maternal to embryonic transition (MET), HP1gamma protein was primarily cytoplasmic, whereas in 8-16-cell embryos (post MET), HP1gamma was primarily nuclear. Lineage-specific patterns of HP1gamma protein localisation become evident from compaction, being restricted to peripheral, extraembryonic cells at the morula and blastocyst stages (Days 7-9). Surprisingly, we detected HP1gamma mRNA in both embryonic and extraembryonic cells in blastocysts by fluorescence in situ hybridisation. In trophectoderm cells, HP1gamma protein was localised in specific patterns at the mitotic and interphase stages of the cell cycle. These results demonstrate lineage- and cell cycle-specific patterns of HP1gamma protein localisation in the post-compaction, preimplantation bovine embryo and raise interesting questions about the role of HP1gamma in early embryo development.

Species-specific differences in the activity and nuclear localization of murine and bovine phospholipase C zeta 1.

Injection of mammalian sperm extracts or cRNA of the sperm-specific phospholipase C zeta 1 (PLCZ1) has been shown to trigger repetitive oscillations in the concentration of free calcium ([Ca(2+)](i)), leading to oocyte activation and embryo development in all mammals studied to date. While PLCZ1 has cross-species activity, it has also been observed that species-specific differences may exist in the frequency and pattern of the resulting [Ca(2+)](i) oscillations following PLCZ1 cRNA injection into oocytes of different species. Accordingly, we used a crossover design strategy to directly investigate the activity of murine and bovine PLCZ1 in both murine and bovine oocytes. In murine oocytes, injection of murine Plcz1 cRNA induced [Ca(2+)](i) oscillations at 10-fold lower concentrations than bovine PLCZ1, although in bovine oocytes bovine PLCZ1 was more effective than murine Plcz1 at inducing [Ca(2+)](i) oscillations. Investigation of ITPR1 (IP(3)R1) down-regulation in bovine oocytes by PLCZ1 cRNA also showed that bovine PLCZ1 was more active in homologous oocytes. To determine whether these PLCZs exhibited similar cellular distribution, Venus-tagged PLCZ1 cRNA was injected into oocytes, and PLCZ1 was overexpressed. Bovine PLCZ1 failed to accumulate in the pronucleus (PN) of bovine or murine zygotes, despite possessing a putative nuclear localization signal. Conversely, murine PLCZ1 accumulated in the PN of both murine and bovine zygotes. These results demonstrate that murine PLCZ1 and bovine PLCZ1 possess species-specific differences in activity and suggest potential differences in the mode of action of the protein between the two species. Variation in sperm PLCZ1 protein content among species, along with oocyte-specific differences in the localization and availability of PLCZ1 substrates, may further contribute to optimize the activation stimulus to enhance embryo development.

Developmental disparity between in vitro-produced and somatic cell nuclear transfer bovine days 14 and 21 embryos: implications for embryonic loss.

In ruminants, the greatest period of embryonic loss coincides with the period of elongation when the embryonic disc is formed and gastrulation occurs prior to implantation. The impact of early embryonic mortality is not only a major obstacle to the cattle breeding industry but also impedes the application of new reproductive technologies such as somatic cell nuclear transfer (SCNT). In the present study, days 14 and 21 bovine embryos, generated by either in vitro-production (IVP) or SCNT, performed by either subzonal injection (SUZI) or handmade cloning (HMC), were compared by stereomicroscopy, immunohistochemistry, and transmission electron microscopy to establish in vivo developmental milestones. Following morphological examination, samples were characterized for the presence of epiblast (POU5F1), mesoderm (VIM), and neuroectoderm (TUBB3). On D14, only 25, 15, and 7% of IVP, SUZI, and HMC embryos were recovered from the embryos transferred respectively, and similar low recovery rates were noted on D21, suggesting that most of the embryonic loss had already occurred by D14. A number of D14 IVP, SUZI, and HMC embryos lacked an epiblast, but presented trophectoderm and hypoblast. When the epiblast was present, POU5F1 staining was limited to this compartment in all types of embryos. At the ultrastructural level, SCNT embryos displayed abundant secondary lysosomes and vacuoles, had fewer mitochondria, polyribosomes, tight junctions, desmosomes, and tonofilaments than their IVP counterparts. The staining of VIM and TUBB3 was less distinct in SCNT embryos when compared with IVP embryos, indicating slower or compromised development. In conclusion, SCNT and to some degree, IVP embryos displayed a high rate of embryonic mortality before D14 and surviving embryos displayed reduced quality with respect to ultrastructural features and differentiation markers.

Dynamic changes in the localization of five members of the methyl binding domain (MBD) gene family during murine and bovine preimplantation embryo development.

There are five methyl binding domain (MBD) proteins characterized by a methyl CpG-binding domain. Four MBD proteins (MeCP2 and MBDs 1-3) are linked to transcriptional repression and one (MBD4), to DNA repair. During preimplantation development, the embryo undergoes global demethylation following fertilization and selective remethylation following the maternal to zygotic transition (MZT). This study characterized changes in MBD mRNA expression and protein localization during both murine and bovine preimplantation development. These species were selected because they undergo MZT at different developmental stages. Gene expression profiling during preimplantation development detected the presence of all MBDs examined, although stage and species-specific differences were observed. MBD2 was not expressed in murine or bovine oocytes and MeCP2 was not detected in murine blastocysts, subcellular protein localization was found to vary at time points critical in development. Most MBDs showed species-specificity in localization patterns and differences were found between individual MBDs. MBD1 localization is consistent with a novel role during MZT for both species. MBD3, known to play a crucial role in murine embryogenesis, was highly localized to the nucleus before and after, but not during the MZT in the bovine. MBD2, MBD4, and MeCP2 show varying patterns of localization which indicate possible roles in the early cleavage stages and in inner cell mass differentiation. Further experiments are currently underway to define discreet functional roles for specific MBDs during bovine preimplantation embryogenesis.

Dynamic changes in localization of Chromobox (Cbx) family members during the maternal to embryonic transition.

The Chromobox domain (Cbx) gene family, consisting of Polycomb and Heterochromatin Protein 1 genes, is involved in transcriptional repression, cell cycle regulation and chromatin remodeling. We report the first study of gene expression and protein localization of the Cbx genes in in vitro produced bovine embryos. All but one gene (Cbx6) were expressed. This was confirmed by immunolocalization for HP1alpha, beta, gamma, and Pc2, 3. HP1beta was found in the nuclei of embryos from the two-cell stage onwards, whereas HP1gamma showed diffuse cytoplasmic/nuclear localization at the two- and eight-cell stages, and predominantly nuclear localization at the four-cell stage and the 16-cell stage onwards. Leptomycin B (LMB), a specific inhibitor of the nuclear export protein CRM-1 (chromosomal regional maintenance-1), was found to increase nuclear localization of HP1gamma at the eight-cell stage, and to prevent progression past this stage of embryogenesis. This indicates that HP1gamma possesses a CRM-1-dependent nuclear export pathway which may represent part of the basis of HP1gamma's ability to shuttle between the nucleus and the cytoplasm in dynamic fashion. HP1alpha was expressed in embryonic nuclei at all stages, but was found to relocalise from euchromatin to heterochromatin during the maternal to embryonic transition (MET). In contrast, Pc2 and Pc3 were evenly distributed between cytoplasm and nucleus until the eight- and sixteen-cell stages or the morula stage, respectively, before relocating preferentially to the cytoplasm. Collectively, the results suggest that dynamic changes of the nuclear-cytoplasmic and subnuclear distribution of members of the Cbx family may be central to the MET.