TFPI from erythroblasts drives heme production in central macrophages promoting erythropoiesis in polycythemia.

Bleeding and thrombosis are known as common complications of polycythemia for a long time. However, the role of coagulation system in erythropoiesis is unclear. Here, we discover that an anticoagulant protein tissue factor pathway inhibitor (TFPI) plays an essential role in erythropoiesis via the control of heme biosynthesis in central macrophages. TFPI levels are elevated in erythroblasts of human erythroblastic islands with JAK2V617F mutation and hypoxia condition. Erythroid lineage-specific knockout TFPI results in impaired erythropoiesis through decreasing ferrochelatase expression and heme biosynthesis in central macrophages. Mechanistically, the TFPI interacts with thrombomodulin to promote the downstream ERK1/2-GATA1 signaling pathway to induce heme biosynthesis in central macrophages. Furthermore, TFPI blockade impairs human erythropoiesis in vitro, and normalizes the erythroid compartment in mice with polycythemia. These results show that erythroblast-derived TFPI plays an important role in the regulation of erythropoiesis and reveal an interplay between erythroblasts and central macrophages.

Identification and characterization of two novel antimicrobial peptides from Japanese sea bass (Lateolabrax japonicus) with antimicrobial activity and MO/MФ activation capability.

Piscidins participate in the innate immune response of fish, which aims to eliminate recognized foreign microbes and restore the homeostasis of immune system. We characterized two piscidin-like antimicrobial peptides (LjPL-3 and LjPL-2) isolated from Japanese sea bass (Lateolabrax japonicus). LjPL-3 and LjPL-2 showed different expression patterns in tissues. After Vibrio harveyi infection, the mRNA expression of LjPL-3 and LjPL-2 was upregulated in the liver, spleen, head kidney, and trunk kidney. The synthetic mature peptides LjPL-3 and LjPL-2 exhibited different antimicrobial spectra. Furthermore, LjPL-3 and LjPL-2 treatments decreased inflammatory cytokine production while promoting chemotaxis and phagocytosis in monocytes/macrophages (MO/MФ). LjPL-2, but not LjPL-3, displayed bacterial killing capability in MO/MФ. LjPL-3 and LjPL-2 administration increased Japanese sea bass survival after V. harveyi challenge, which was accompanied by a decline in bacterial burden. These data suggested that LjPL-3 and LjPL-2 participate in immune response through direct bacterial killing and MO/MФ activation.

Functional and hepatic metabolite changes in aquatic turtle hatchlings exposed to the anti-androgenic fungicide vinclozolin.

Many man-made chemicals that are released into water bodies in agricultural landscapes have been identified as endocrine disruptors and can cause serious impacts on the growth and survival of aquatic species living in these environments. However, very little attention has been paid to their toxicological effects in cultured non-fish species, such as aquatic turtles. We exposed hatchlings of the Chinese soft-shelled turtle (Pelodiscus sinensis) to different concentrations of vinclozolin (0, 5, 50 and 500 µg/L) for 60 days to assess physiological and metabolic impacts of this fungicide. Despite no death occurrence, hatchling turtles exposed to the highest concentration of vinclozolin consumed less food, grew more slowly (resulting in smaller body size after exposure) and performed more poorly in behavioral swimming tests than controls and turtles exposed to lower concentrations. Hepatic metabolite profiles acquired via liquid chromatography-mass spectrometry (LC-MS) revealed multiple metabolic perturbations related to amino acid, lipid, and fatty acid metabolism in animals exposed to environmentally relevant concentrations. Specifically, many critical metabolites involved in energy-related metabolic pathways (such as some intermediates in the tricarboxylic acid cycle, lactate, and some amino acids) were present in livers of hatchling turtles exposed vinclozolin, though at lower concentrations, reflecting energy metabolism dysregulation induced by exposure to this fungicide. Overall, our results suggest that the changes in growth and behavioral performances caused by chronic vinclozolin exposure may be associated with internal physiological and metabolic disorders mediated at the biochemical level.

Molecular cloning, the characterization of metallothionein and catalase, and the evaluation of testicular toxicity of Cd in the Chinese fire-bellied newt (Cynops orientalis).

Cadmium (Cd) is an environmental toxicant and a nonessential metal. Cd can attack a wide range of organs, such as the liver, kidney, lung, ovary, testis, brain, and muscle in vertebrates. Among these organs, the testis might be the most sensitive organ to Cd toxicity. Metallothionein (MT) is a cysteine-rich protein with a low molecular weight, that can bind with Cd and eliminate reactive oxygen species (ROSs). Hydrogen peroxide, which as a crucial type of ROS that is induced by Cd, can be eliminated by catalase (CAT) in the self-protection of cells and to realize Cd toxicity resistance. To investigate the functions of MT and CAT in the testis of Cynops orientalis, we cloned the full-length MT and CAT genes of C. orientalis for the first time. Immunofluorescence results demonstrated that MT and CAT were expressed in Sertoli cells and all spermatogenic cells in the testis of C. orientalis. The results of the ultrastructural damage assay demonstrated that there were various impairments, which included organelle vacuolization, abnormal chromatin distribution, and apoptotic bodies, in somatic cells that were exposed to Cd. However, the anomalies of spermatozoa were located mainly in the mid-piece and head, many of which showed severely impaired structures. The results demonstrated that MT and CAT expression had distinct patterns in response to various Cd concentrations: an increase in MT mRNA levels with elevated Cd levels and a persistent increase in CAT mRNA levels with elevated Cd levels. These results suggested that MT and CAT play roles in Cd toxicity resistance in the testis and that the expression of CAT may be a better biomarker than the expression of MT for assessing Cd pollution.

Gene expression pattern of KIFC3 during spermatogenesis of the skink Eumeces chinensis.

Kinesin superfamily is a class of microtubule-dependent motors that play crucial roles in acrosome biogenesis, nuclear reshaping and flagellum formation during spermiogenesis. We have cloned kinesin-like gene kifc3 (termed ec-kifc3) from the total RNA of the testis of the skink Eumeces chinensis. The cDNA sequence of ec-kifc3 had a full-length of 3033bp, including a 260bp 5'-untranslated region (5'UTR), a 445bp 3'-untranslated region (3'UTR) and an open reading frame that encoded a 775-amino-acid protein. Additionally, the calculated molecular weight of the putative ec-KIFC3 was 87kDa and its estimated isoelectric point was 6.18. Structurally, the putative ec-KIFC3 had three domains: head domain, neck domain and tail domain. Protein alignment demonstrated that ec-KIFC3 had 47.2%, 67.8%, 68.8%, 69.3% and 76.8% identity with its homologues in Xenopus laevis, Mus musculus, Cricetulus griseus, Homo sapiens, and Gallus gallus. The phylogenetic analysis showed that ec-KIFC3 was more related to KIFC3 in vertebrates than invertebrates. Tissue expression results showed the presence of ec-KIFC3 in various tissues with its highest expression in the testis. In situ hybridization demonstrated that ec-KIFC3 mRNA was distributed around the nucleus in early and middle stage spermatids and expressed in the nucleus in the elongating spermatids during spermiogenesis. Besides, the ec-KIFC3 mRNA was expressed in the acrosome of the developmental spermatids. From the results of in situ hybridization and previous researches, we speculated that ec-KIFC3 may play a role in nuclear morphogenesis and acrosome formation during spermiogenesis of E. chinensis.

Molecular characterization and expression analysis of a KIFC1-like kinesin gene in the testis of Eumeces chinensis.

The member of the kinesin-14 subfamily, KIFC1, is a carboxyl-terminal motor protein that plays an important role in the elongation of nucleus and acrosome biogenesis during the spermiogenesis of mammals. Here, we had cloned and sequenced the cDNA of a mammalian KIFC1 homologue (termed ec-KIFC1) from the total RNA of the testis of the reptile Eumeces chinensis. The full-length sequence was 2,339 bp that contained a 216 bp 5'-untranslated region (5'UTR), a 194 bp 3'-untranslated region (3'UTR) and a 1,929 bp open reading frame that encoded a special protein of 643 amino acids (aa). The calculated molecular weight of the putative ec-KIFC1 was 71 kDa and its estimated isoelectric point was 9.47. The putative ec-KIFC1 protein owns a tail domain from 1 to 116 aa, a stalk domain from 117 to 291 aa and a conserved carboxyl motor domain from 292 to 642 aa. Protein alignment demonstrated that ec-KIFC1 had 45.6, 42.8, 44.6, 36.9, 43.7, 46.4, 45.1, 55.6 and 49.8 % identity with its homologues in Mus musculus, Salmo salar, Danio rerio, Eriocheir sinensis, Rattus norvegicus, Homo sapiens, Bos taurus, Gallus gallus and Xenopus laevis, respectively. Tissue expression analysis showed the presence of ovary, heart, liver, intestine, oviduct, testis and muscle. The phylogenetic tree revealed that ec-KIFC1 was more closely related to vertebrate KIFC1 than to invertebrate KIFC1. In situ hybridization showed that the ec-KIFC1 mRNA was localized in the periphery of the nuclear membrane and the center of the nucleus in early spermatids. In mid spermatids, the ec-KIFC1 had abundant expression in the center of nucleus, and was expressed in the tail and the anterior part of spermatids. In the late spermatid, the nucleus gradually became elongated, and the ec-KIFC1 mRNA signal was still centralized in the nucleus. In mature spermatids, the signal of the ec-KIFC1 gradually became weak, and was mainly located at the tail of spermatids. Therefore, the ec-KIFC1 probably plays a critical role in the spermatogenesis of E. chinensis.

The apoptotic function analysis of p53, Apaf1, Caspase3 and Caspase7 during the spermatogenesis of the Chinese fire-bellied newt Cynops orientalis.

BACKGROUND: Spontaneous and stress-induced germ cell apoptosis during spermatogenesis of multicellular organisms have been investigated broadly in mammals. Spermatogenetic process in urodele amphibians was essentially like that in mammals in spite of morphological differences; however, the mechanism of germ cell apoptosis in urodele amphibians remains unknown. The Chinese fire-belly newt, Cynops orientalis, was an excellent organism for studying germ cell apoptosis due to its sensitiveness to temperature, strong endurance of starvation, and sensitive skin to heavy metal exposure. METHODOLOGY/PRINCIPAL FINDINGS: TUNEL result showed that spontaneous germ cell apoptosis took place in normal newt, and severe stress-induced apoptosis occurred to spermatids and sperm in response to heat shock (40°C 2 h), cold exposure (4°C 12 h), cadmium exposure (Cd 36 h), and starvation stress. Quantitative reverse transcription polymerase chain reactions (qRT-PCR) showed that gene expression of Caspase3 or Caspase7 was obviously elevated after stress treatment. Apaf1 was not altered at its gene expression level, and p53 was significantly decreased after various stress treatment. Caspase assay demonstrated that Caspase-3, -8, -9 enzyme activities in newt testis were significantly elevated after heat shock (40°C 2 h), cold exposure (4°C 12 h), and cadmium exposure (Cd 36 h), while Caspase3 and Caspase8 activities were increased with Caspase9 significantly decreased after starvation treatment. CONCLUSIONS/SIGNIFICANCE: Severe germ cell apoptosis triggered by heat shock, cold exposure, and cadmium exposure was Caspase3 dependent, which probably involved both extrinsic and intrinsic pathways. Apaf1 may be involved in this process without elevating its gene expression. But starvation-induced germ cell apoptosis was likely mainly through extrinsic pathway. p53 was probably not responsible for stress-induced germ cell apoptosis in newt testis. The intriguing high occurrence of spermatid and sperm apoptosis probably resulted from the sperm morphology and unique reproduction policy of Chinese fire-belly newt, Cynops orientalis.

Molecular characterization of a KIF3A-like kinesin gene in the testis of the Chinese fire-bellied newt Cynops orientalis.

KIF3A, the subunit within the kinesin-2 superfamily, is a typically N-terminal motor protein, which is involved in membranous organelle and intraflagellar transport. During spermatogenesis, KIF3A plays a critical role in the formation of flagella and cilia. KIF3A is also related to the left-right asymmetry, the signal pathway, DNA damage and tumorigenesis. We used RT-PCR and in situ hybridization to clone the kif3a gene, and we identified its function in the testis of the Chinese fire-bellied newt Cynops orientalis (termed as co-kif3a). The full-length sequence of co-kif3a was 2193 bp, containing a 56 bp 5'UTR, 2073 bp ORF encoding a protein of 691 amino acids and a 64 bp 3'UTR. The secondary structure analysis showed that co-KIF3A had three motor domains, representing the N-terminal motor domain (1-400 aa), α-helix domain (400-600 aa) and C-terminal tail domain (600-691 aa). The amino acid sequence of co-KIF3A shared an identity of 55.9%, 90.9%, 89.9%, 91.3% and 85.7% with its counterparts in Aedes aegypti, Mus musculus, Xenopus tropicalis, Homo sapiens and Danio rerio, respectively. The calculated molecular weight of the putative co-KIF3A was 79 kDa and its estimated isoelectric point was 6.8. RT-PCR result showed that co-kif3a was expressed in several examined tissues, with a high level in the testis and low levels in liver, muscle and ovum. Kif3a was weakly expressed in the heart and spleen, and barely detected in the intestine. In situ hybridization analysis demonstrated that in early spermatid co-kif3a was expressed around the nuclear membrane. When the tail began to emerge in the middle spermatid, mRNA transcript was abundantly concentrated in the flagellum. The mRNA signal was still very strong along all the flagellum in late spermatid. In mature spermatid, the message was weak. Therefore, co-KIF3A probably plays a functional role in the spermiogenesis of C. orientalis.

[The effects of hypoxia during pregnancy on sexual behavior of male offspring].

OBJECTIVE: To investigate the effects of hypoxia during the prenatal period and its later repercussions on sexual behavior and the sex hormone secretion of male rats. METHODS: Experimental animals were divided into three groups randomly: control group, which was kept at normal atmospheric pressure, and two stress groups exposed to a simulated altitude equivalent to 3000 m and 5000 m, respectively. Stress groups were exposed to hypoxic circumstance at their final week of gestation in animal decompression chamber. RESULTS: Adulthood, males exposed to hypoxic stress during the prenatal period were able to mate with normal females, but these treated males exhibited decreased male sexual behavior. Decreased anogenital distances were observed in male offspring, and presented reductions of plasma testosterone levels, increase of plasma corticosterone levels, but no notable alteration in the organ index. CONCLUSION: These results indicate that exposure to hypoxia in the later stages of pregnancy may have a long-term effect on the fertility and sexual behavior of male offspring.

[Establishment of loop-mediated isothermal amplification for detection of Vibrio alginolyticus].

Vibrio alginolyticus is the main Vibrio pathogen in aquaculture in the south of China. A one step loop-mediated isothermal amplification (LAMP) assay was developed for detection of Vibrio alginolyticus. A set of primers were designed from the OmpK sequence of Vibrio alginolyticus. The assay was optimised to amplify Vibrio alginolyticus DNA by incubation at 65 degrees C for only 1 h, and required only a simple water bath or heating block to provide a constant temperature of 65 degrees C. LAMP amplification products had a ladder-like appearance when electrophoresed on an agarose gel. The detection limit of the LAMP assay was n(cell) = 38/mL which was found to be higher than the commonly used PCR method. The assay was evaluated using clinical samples and the results indicated the suitability and simplicity of the test as a rapid, field diagnostic tool for Vibrio alginolyticus.

[Pattern of plasma sex steroid hormone levels during the breeding season of male and female skink: Eumeces chinensis].

Changes in gonadal activity and plasma sex steroid hormone levels in male and female Eumece chinensis during the breeding season were described. The results showed that: The vitellogensis of follicles of female Eumeces chinensis needed the stimulation of 17beta-estradiol (E2). As ovary masses reached peak values between late April and mid-May, E2 levels rose to the top value by late March, and then sharply declined but went up again before preovulation; The physiological functions of plasma progesterone (P) consisted in its oviductal egg retention, embryo development, and eggshell formation. P levels fluctuated near the basic value between mid-March and late April. In mid-May, with the onset of ovulation, plasma P levels rose rapidly, reached peak value by late May and declined sharply after ovulation. Plasma E2 levels declined as plasma P levels rose, showing an inverse relationship between them; In males, plasma Testosterone (T) levels were closely correlated with the maintenance of spermatogenesis activities, male and male combat, sexual display, and mating. Plasma T levels tended to rise after the termination of hibernation, and reached peak value by mid-April. After mid-May, with the testis aggressing, plasma T levels gradually went down and reached bottom value by late June.