Epidermal growth factor: Expression in goat endometrial epithelia during early pregnancy and regulation by interferon tau and FOXO1.

In ruminants, establishment and maintenance of pregnancy depends upon a well-coordinated interaction between the conceptus and the maternal endometrium. Epidermal growth factor (EGF) is important for embryo implantation and pregnancy establishment. However, the regulatory mechanisms of EGF expression remain unclear. FOXO1, a member of the Forkhead box O (FOXO) subfamily of transcription factors, is currently accepted as a novel endometrial receptivity marker for humans and mice owing to its timely and specific expression at the window of implantation. In this study, we examined the spatiotemporal expression profile of EGF in goat uterus during early pregnancy (Day 0 to Day 50 of pregnancy) and verified that EGF expression was regulated by FOXO1 and interferon tau (IFNT). Our results showed that EGF was highly expressed in the luminal epithelium (LE) and the glandular epithelium (GE) during conceptus adhesion (Day 16 to Day 25 of pregnancy). After implantation, EGF protein signals were continuously detected in the endometrial epithelia and appeared in the conceptus trophectoderm. Furthermore, EGF expression could be up-regulated by IFNT in goat uterus and primary endometrial epithelium cells (EECs). The luciferase assay results showed that FOXO1 could promote EGF transcription by binding to its promoter. And FOXO1 positively regulates EGF expression in goat EECs. These findings contribute to better understanding the role and regulation mechanisms of EGF during ruminant early pregnancy.

Production of all-male non-transgenic zebrafish by conditional primordial germ cell ablation.

Many fish species exhibit remarkable sexual dimorphism, with males possessing numerous advantageous traits for commercial production by aquaculture such as faster growth rate, more efficient food energy utilization for muscle development, and better breeding performance. Several studies have shown that a decrease in the number of primordial germ cells (PGCs) during early development leads predominantly to male progeny. In this study, we developed a method to obtain all-male zebrafish (Danio rerio) by targeted PGC ablation using the nitroreductase/metronidazole (NTR/Mtz) system. Embryos generated by female heterozygous Tg(nanos3:nfsB-mCherry-nanos3 3'UTR) and male wild-types (WTs) were treated with vehicle or Mtz. Compared to vehicle-treated controls, 5.0 and 10.0 mM Mtz treatment for 24 h significantly reduced the number of PGCs and yielded an exclusively male phenotype in adulthood. The gonads of offspring treated with 5.0 mM Mtz exhibited relatively normal morphology and histological characteristics. Furthermore, these males were able to chase females, spawn, and produce viable offspring, while about 20.0% of males treated with 10.0 mM Mtz were unable to produce viable offspring. The 5.0 mM Mtz treatment protocol may thus be suitable for large-scale production of fertile male offspring. Moreover, about half of these males were WT as evidenced by the absence of nfsB gene expression. It may thus be possible to breed an all-male WT fish population by Mtz-mediated PGC ablation.

METTL3 promotes proliferation of goat endometrial epithelial cells by regulating CTGF in an m6A-dependent manner†.

N6-methyladenosine (m6A), an epigenetic modification on RNAs, plays an important role in many physiological and pathological processes. However, the involvement of m6A in goat uterus during early pregnancy remains largely unknown. In this study, we found that the total m6A level was increasing in goat uterus as early pregnancy progressed. Methyltransferase-like 3 (METTL3) is a core catalytic subunit of the m6A methyltransferase. We thus determined the expression and regulation of METTL3 in goat uterus. METTL3 was highly expressed in the luminal and glandular epithelia from day 16 (D16) to D25 of pregnancy, and it could be up-regulated by estrogen and progesterone in goat uterus and primary endometrial epithelial cells (EECs). In EECs, knockdown or overexpression of METTL3 resulted in a significant decrease or increase of cell proliferation, respectively. METTL3 knockdown reduced the m6A level of not only total RNA but also connective tissue growth factor (CTGF) mRNA. Luciferase assay suggested that METTL3 might target the potential m6A sites in the 3'untranslated region (3'UTR) of CTGF mRNA. Moreover, METTL3 positively regulated CTGF expression, and CTGF knockdown significantly counteracted the promoting effect of METTL3 overexpression on EEC proliferation. Collectively, METTL3 is dynamically expressed in goat uterus and can affect EEC proliferation by regulating CTGF in an m6A-dependent manner. Our results will lay a foundation for further studying the crucial mechanism of METTL3-mediated m6A modification in goat uterus during early pregnancy.

Multiple paternity in the cultured yellow pond turtles (Mauremys mutica).

As a result of hunting and habitat loss, wild populations of the yellow pond turtle, Mauremys mutica, are decreasing. The International Union for Conservation of Nature considers M. mutica to be an endangered species. All studied freshwater turtles have polyandrous mating with multiple paternity. To survey the mating strategies of M. mutica, 1year's genetic data of parents and all offspring in an artificially captive population were analyzed. Two groups of multiplex PCR containing 16 microsatellite loci were used to analyze the paternity of 302 hatchlings from 132 parents and from 159 clutches. The genetic data indicated that multiple paternity is rare in M. mutica, occurring in only seven of 138 clutches. Although the frequency of multiple paternity was only 5.07%, results of the present research indicate that M. mutica has a polyandrous mating system. In the breeding season, the successive clutches of 34 females each had the same paternity as the previous clutches. It was observed that four males (f85, f58, f87, and f76) had more than 20 offspring each, totaling 99 and representing 32.78% of all offspring. This finding implies that paternity is competitive in this artificially captive population and might bias the genetic diversity of the offspring.

Novel duplication pattern of the mitochondrial control region in Cantor's Giant softshell turtle Pelochelys cantorii.

Cantor's Giant Softshell Turtle, Pelochelys cantorii has become one of the most critically endangered species in the world. When comparative analyses of the P. cantorii complete mitochondrial genome sequences were conducted, we discovered a duplication of a segment of the control region in the mitochondrial genome of P. cantorii. The duplication is characterized by two copies of conserved sequence box 2 (CSB2) and CSB3 in a single control region. In contrast to previous reports of duplications involving the control regions of other animals, this particular pattern of duplications appears to be unique to P. cantorii. Copies of the CSB2 and CSB3 show many of the conserved sequence features typically found in mitochondrial control regions, and rare differences were found between the paralogous copies. Using the primer design principle of simple sequence repeats (SSR) and the reference sequence of the duplicated CSBs, specific primers were designed to amplify the duplicated CSBs. These primers were validated among different individuals and populations of P. cantorii. This unique duplication structure suggests the two copies of the CSB2 and CSB3 may have arisen through occasional tandem duplication and subsequent concerted evolution.

Complete mitochondrial genome of Cuora trifasciata (Chinese three-striped box turtle), and a comparative analysis with other box turtles.

Cuora trifasciata has become one of the most critically endangered species in the world. The complete mitochondrial genome of C. trifasciata (Chinese three-striped box turtle) was determined in this study. Its mitochondrial genome is a 16,575-bp-long circular molecule that consists of 37 genes that are typically found in other vertebrates. And the basic characteristics of the C. trifasciata mitochondrial genome were also determined. Moreover, a comparison of C. trifasciata with Cuora cyclornata, Cuora pani and Cuora aurocapitata indicated that the four mitogenomics differed in length, codons, overlaps, 13 protein-coding genes (PCGs), ND3, rRNA genes, control region, and other aspects. Phylogenetic analysis with Bayesian inference and maximum likelihood based on 12 protein-coding genes of the genus Cuora indicated the phylogenetic position of C. trifasciata within Cuora. The phylogenetic analysis also showed that C. trifasciata from Vietnam and China formed separate monophyletic clades with different Cuora species. The results of nucleotide base compositions, protein-coding genes and phylogenetic analysis showed that C. trifasciata from these two countries may represent different Cuora species.

The complete mitochondrial genome of the critically endangered Vietnamese three-striped box turtle (Testudines: Geoemydidae).

The complete mitochondrial genome of the Vietnamese three-striped box turtle (Cuora cyclornata) was first determined in this study. It was a circular molecule of 16,594 bp in length, consisting of 37 genes typically found in other vertebrates. The AT content of the overall base composition of the whole mitogenome was 60.39%, while the control region was 70.23%. Two ETAS and 4 CSBs were identified, while a remarkable feature was found in the control region: a large number of (TTATTATA)10 direct tandem repeats followed by (TTATA)n (n=10, 8 and 1), which were spaced into three domains by (TA)n (n=1, 1 and 2). The sequence information could play an important role in the study of phylogenetic relationships in turtles and preservation of genetic resources for helping conservation of the endangered species.

Variation in copper and zinc tolerance and accumulation in 12 willow clones: implications for phytoextraction.

Willows (Salix spp.) have shown high potential for the phytoextraction of heavy metals. This study compares variations in copper (Cu) and zinc (Zn) tolerance and accumulation potential among 12 willow clones grown in a nutrient solution treated with 50 µmol/L of Cu or Zn, respectively. The results showed differences in the tolerance and accumulation of Cu and Zn with respect to different species/clones. The biomass variation among clones in response to Cu or Zn exposure ranged from the stimulation of growth to inhibition, and all of the clones tested showed higher tolerance to Cu than to Zn. The clones exhibited less variation in Cu accumulation but larger variation in Zn accumulation. Based on translocation factors, it was found that most of the Cu was retained in the roots and that Zn was more mobile than Cu for all clones. It is concluded that most willow clones are good accumulators of Zn and Cu.

[Cloning and sequencing of the GTP cyclohydrolase I function region of azole target enzyme gene in artificially induced resistant Candida albicans].

OBJECTIVE: To study the relationship between gene mutation of GTP cyclohydrolase I function region in lanosterine 14 alpha-demethylase (14-DM) DNA sequence and drug resistance of Candida albicans. METHODS: One standard strain and 2 isolate strains of Candida albicans were induced artificially by fluconazole plus albendazole. The gene fragments of the 3 strains and another 2 clinical isolates which were resistant to fluconazol were detected by PCR, and then cloned onto pMD-18T vectors to sequence and analyze the change of gene sequence after the induction. RESULTS: The sequences underwent substantial gene mutations after induction. Some of the mutations resulted in alteration of amino acids. The sequence change and subsequent alteration of amino acids in the tested strains coincided with those in the clinical isolates. CONCLUSION: Gene mutation and alteration of amino acid of 14-DM GTP domain are related to azole-resistance in Candida albicans.