Molecular characterization of Lhx8, an oocyte-specific transcription factor, and its interacting proteins in rainbow trout and cattle

LIM homeobox 8 (Lhx8) is an important transcription factor that is preferentially expressed in germ cells. Lhx8 null mice are infertile due to lack of oocytes and impairment of the transition from primordial follicles to primary follicles. Lhx8 deficiency also affects the expression of many important oocyte-specific genes. To date, no attempts have been made to investigate the existence of any cellular factors that might interact with Lhx8 protein in oocytes and early embryos. In this study, we report the characterization of rainbow trout and bovine Lhx8 genes and identification of important germ cell-specific nuclear factors that interact with Lhx8 protein in both species. In rainbow trout, two Lhx8 genes, Lhx8a and Lhx8b, were identified, encoding proteins of 344 and 361 amino acids, respectively. The two proteins share 83% sequence identity and both transcripts are specifically expressed in the ovary. Quantitative real time PCR analysis demonstrated that both genes are expressed highly in pre-vitellogenic ovaries as well as in early stage embryos. Using a yeast two-hybrid screening system, a novel protein (Borealin-2) interacting with Lhx8 was identified. The interaction between either Lhx8a or Lhx8b and Borealin-2 was further confirmed by a bimolecular fluorescence complementation (BiFC) assay. Borealin-2 is a protein of 255 amino acids containing an Nbl1 domain, and its mRNA expression is restricted to the ovary and testis. A GFP reporter assay revealed that Borealin-2 is a nuclear protein. Results indicate that both Lhx8a and Lhx8b function through interaction with Borealin-2, which may play an important role during oogenesis and early embryogenesis in rainbow trout. The open reading frame (ORF) of bovine Lhx8 gene was amplified from cDNA of a bovine fetal ovary using primers designed based on a predicted bovine Lhx8 cDNA sequence and a partial 5 \u27end transcript. The ORF of bovine Lhx8 cDNA is 1,134 bp in length encoding a protein of 377 amino acids. A splicing variant of Lhx8 (Lhx8_v1) was identified, which results from alternative splicing of exon 2 and 3, and encodes a protein of 293 amino acids. The predicted bovine Lhx8 protein contains two LIM domains and one homeobox domain. However, one of the LIM domains in the splicing variant, Lhx8_v1, is incomplete due to deletion of 83 amino acids near the N terminus. Both Lhx8 and Lhx8_v1 mRNA are specifically expressed in fetal ovaries and testis but not detectable in the somatic tissues as well as in granulosa and theca cells. Lhx8 mRNA is highly abundant in GV and MII stage oocytes as well as in early stage embryos but not detectable in morula and blastocyst stage embryos. Lhx8_v1 mRNA expression is detectable in oocytes and early embryo but not in morula and blastocyst stage embryos. A GFP reporter assay revealed that Lhx8 is a nuclear protein and the predicted monopartite NLS is required for its transport into the nucleus. Direct yeast two-hybrid analysis revealed that bovine Lhx8 protein interacts with Figla, a basic helix-loop-helix transcription factor. The interaction between Lhx8 and Figla was confirmed by a co-immunoprecipitation assay. This is the first time that a direct protein-protein interaction between two germ cell-specific transcription factors essential for oocyte and follicular development is demonstrated. The study provides new information for studying the mechanisms of the regulatory roles of Lhx8 in oocyte/follicular development and early embryogenesis

Bovine Lhx8, a Germ Cell-SpecificNuclear Factor, Interacts with Figla

LIM homeobox 8 (Lhx8) is a germ cell-specific transcription factor essential for the development of oocytes during early oogenesis. In mice, Lhx8 deficiency causes postnatal oocyte loss and affects the expression of many oocyte-specific genes. The aims of this study were to characterize the bovine Lhx8 gene, determine its mRNA expression during oocyte development and early embryogenesis, and evaluate its interactions with other oocyte-specific transcription factors. The bovine Lhx8 gene encodes a protein of 377 amino acids. A splice variant of Lhx8 (Lhx8_v1) was also identified. The predicted bovine Lhx8 protein contains two LIM domains and one homeobox domain. However, one of the LIM domains in Lhx8_v1 is incomplete due to deletion of 83 amino acids near the N terminus. Both Lhx8 and Lhx8_v1 transcripts were only detected in the gonads but none of the somatic tissues examined. The expression of Lhx8 and Lhx8_v1 appears to be restricted to oocytes as none of the transcripts was detectable in granulosa or theca cells. The maternal Lhx8 transcript is abundant in GV and MII stage oocytes as well as in early embryos but disappear by morula stage. A nuclear localization signal that is required for the import of Lhx8 into nucleus was identified, and Lhx8 is predominantly localized in the nucleus when ectopically expressed in mammalian cells. Finally, a novel interaction between Lhx8 and Figla, another transcription factor essential for oogenesis, was detected. The results provide new information for studying the mechanisms of action for Lhx8 in oocyte development and early embryogenesis

DNA Methylation and miRNA-1296 Act in Concert to Mediate Spatiotemporal Expression of KPNA7 During Bovine Oocyte and Early Embryonic Development

Abstract Background: Epigenetic regulation of oocyte-specific maternal factors is essential for oocyte and early embryonic development. KPNA7 is an oocyte-specific maternal factor, which controls transportation of nuclear proteins important for early embryonic development. To elucidate the epigenetic mechanisms involved in the controlled expression of KPNA7, both DNA methylation associated transcriptional silencing and microRNA (miRNA)-mediated mRNA degradation of KPNA7 were examined. Results: Comparison of DNA methylation profiles at the proximal promoter of KPNA7 gene between oocyte and 6 different somatic tissues identified 3 oocyte-specific differentially methylated CpG sites. Expression of KPNA7 mRNA was reintroduced in bovine kidney-derived CCL2 cells after treatment with the methylation inhibitor, 5-aza-2′- deoxycytidine (5-Aza-CdR). Analysis of the promoter region of KPNA7 gene in CCL2 cells treated with 5-Aza-CdR showed a lighter methylation rate in all the CpG sites. Bioinformatic analysis predicted 4 miRNA-1296 binding sites in the coding region of KPNA7 mRNA. Ectopic co-expression of miRNA-1296 and KPNA7 in HEK293 cells led to reduced expression of KPNA7 protein. Quantitative real time PCR (RT-qPCR) analysis revealed that miRNA-1296 is expressed in oocytes and early stage embryos, and the expression reaches a peak level in 8-cell stage embryos, coincident with the time of embryonic genome activation and the start of declining of KPNA7 expression. Conclusions: These results suggest that DNA methylation may account for oocyte-specific expression of KPNA7, and miRNA-1296 targeting the coding region of KPNA7 is a potential mechanism for KPNA7 transcript degradation during the maternal-to-zygotic transition

Lhx8 interacts with a novel germ cell-specific nuclear factor containing an Nbl1 domain in rainbow trout (Oncorhynchus mykiss)

Lhx8 is an important transcription factor that is preferentially expressed in germ cells. Lhx8 null mice are infertile due to lack of oocytes and impairment of the transition from primordial follicles to primary follicles. Lhx8 deficiency also affects the expression of many important oocyte-specific genes. In this study, we report the characterization of rainbow trout lhx8 genes and identification of a novel germ cell-specific nuclear factor that interacts with Lhx8. Two lhx8genes, lhx8a and lhx8b, were identified, encoding proteins of 344 and 361 amino acids, respectively. The two proteins share 83% sequence identity and both transcripts are specifically expressed in the ovary. Quantitative real time PCR analysis demonstrated that both genes are expressed highly in pre-vitellogenic ovaries as well as in early stage embryos. Using a yeast two-hybrid screening system, a novel protein (Borealin-2) interacting with Lhx8 was identified. The interaction between either Lhx8a or Lhx8b and Borealin-2 was further confirmed by a bimolecular fluorescence complementation (BiFC) assay. Borealin-2 is a protein of 255 amino acids containing an Nbl1 domain, and its mRNA expression is restricted to the ovary and testis. A GFP reporter assay revealed that Borealin-2 is a nuclear protein. Collectively, results indicate that both Lhx8a and Lhx8b function through interaction with Borealin-2, which may play an important role during oogenesis and early embryogenesis in rainbow trout

Observation of a thermoelectric Hall plateau in the extreme quantum limit

The thermoelectric Hall effect is the generation of a transverse heat current upon applying an electric field in the presence of a magnetic field. Here we demonstrate that the thermoelectric Hall conductivity $\alpha_{xy}$ in the three-dimensional Dirac semimetal ZrTe$_5$ acquires a robust plateau in the extreme quantum limit of magnetic field. The plateau value is independent of the field strength, disorder strength, carrier concentration, or carrier sign. We explain this plateau theoretically and show that it is a unique signature of three-dimensional Dirac or Weyl electrons in the extreme quantum limit. We further find that other thermoelectric coefficients, such as the thermopower and Nernst coefficient, are greatly enhanced over their zero-field values even at relatively low fields.Comment: 17+21 pages, 3+14 figures; published versio

Temperature- and field angular-dependent helical spin period characterized by magnetic dynamics in a chiral helimagnet MnNb3S6MnNb_3S_6

The chiral magnets with topological spin textures provide a rare platform to explore topology and magnetism for potential application implementation. Here, we study the magnetic dynamics of several spin configurations on the monoaxial chiral magnetic crystal $MnNb_3S_6$ via broadband ferromagnetic resonance (FMR) technique at cryogenic temperature. In the high-field forced ferromagnetic state (FFM) regime, the obtained frequency f vs. resonance field Hres dispersion curve follows the well-known Kittel formula for a single FFM, while in the low-field chiral magnetic soliton lattice (CSL) regime, the dependence of Hres on magnetic field angle can be well-described by our modified Kittel formula including the mixture of a helical spin segment and the FFM phase. Furthermore, compared to the sophisticated Lorentz micrograph technique, the observed magnetic dynamics corresponding to different spin configurations allow us to obtain temperature- and field-dependent proportion of helical spin texture and helical spin period ratio L(H)/L(0) via our modified Kittel formula. Our results demonstrated that field- and temperature-dependent nontrivial magnetic structures and corresponding distinct spin dynamics in chiral magnets can be an alternative and efficient approach to uncovering and controlling nontrivial topological magnetic dynamics.Comment: 29 pages (including Supporting Information), 7 figures, accepted by SCIENCE CHINA Physics, Mechanics & Astronom

Maximizing spin-orbit torque efficiency of Ta(O)/Py via modulating oxygen-induced interface orbital hybridization

Spin-orbit torques due to interfacial Rashba and spin Hall effects have been widely considered as a potentially more efficient approach than the conventional spin-transfer torque to control the magnetization of ferromagnets. We report a comprehensive study of spin-orbit torque efficiency in Ta(O)/Ni81Fe19 bilayers by tuning low-oxidation of \b{eta}-phase tantalum, and find that the spin Hall angle {\theta}DL increases from ~ -0.18 of the pure Ta/Py to the maximum value ~ -0.30 of Ta(O)/Py with 7.8% oxidation. Furthermore, we distinguish the efficiency of the spin-orbit torque generated by the bulk spin Hall effect and by interfacial Rashba effect, respectively, via a series of Py/Cu(0-2 nm)/Ta(O) control experiments. The latter has more than twofold enhancement, and even more significant than that of the former at the optimum oxidation level. Our results indicate that 65% enhancement of the efficiency should be related to the modulation of the interfacial Rashba-like spin-orbit torque due to oxygen-induced orbital hybridization cross the interface. Our results suggest that the modulation of interfacial coupling via oxygen-induced orbital hybridization can be an alternative method to boost the change-spin conversion rate.Comment: 15 pages, 4 figure

Highly Efficient Decomposition of Perfluorocarbons for over 1000 Hours via Active Site Regeneration

Tetrafluoromethane (CF4), the simplest perfluorocarbon (PFC), has the potential to exacerbate global warming. Catalytic hydrolysis is a viable method to degrade CF4, but fluorine poisoning severely restricts both the catalytic performance and catalyst lifetime. In this study, Ga is introduced to effectively assists the defluorination of poisoned Al active sites, leading to highly efficient CF4 decomposition at 600 °C with a catalytic lifetime exceeding 1,000 hours. 27Al and 71Ga magic-angle spinning nuclear magnetic resonance spectroscopy (MAS NMR) showed that the introduced Ga exists as tetracoordinated Ga sites (GaIV), which readily dissociate water to form Ga−OH. In situ diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) and density function theory (DFT) calculations confirmed that Ga−OH assists the defluorination of poisoned Al active sites via a dehydration-like process. As a result, the Ga/Al2O3 catalyst achieved 100 % CF4 decomposition keeping an ultra-long catalytic lifetime and outperforming reported results. This work proposes a new approach for efficient and long-term CF4 decomposition by promoting the regeneration of active sites