Molecular evolution of coding and non-coding sequences of the growth hormone receptor (GHR) gene in the family Bovidae

The GHR gene exon 1A and exon 4 with fragments of its flanking introns were sequenced in twelve Bovidae species and the obtained sequences were aligned and analysed by the ClustalW method. In coding exon 4 only three interspecies differences were found, one of which had an effect on the amino-acid sequence--leucine 152 proline. The average mutation frequency in non-coding exon 1A was 10.5 per 100 bp, and was 4.6-fold higher than that in coding exon 4 (2.3 per 100 bp). The mutation frequency in intron sequences was similar to that in non-coding exon 1A (8.9 vs 10.5/100 bp). For non-coding exon 1A, the mutation levels were lower within than between the subfamilies Bovinae and Caprinae. Exon 4 was 100% identical within the genera Ovis, Capra, Bison, and Bos and 97.7% identical for Ovis moschatus, Ammotragus lervia and Bovinae species. The identity level of non-coding exon 1A of the GHR gene was 93.8% between species belonging to Bovinae and Caprinae. The average mutation rate was 0.2222/100 bp/MY and 0.0513/100 bp/MY for the Bovidae GHR gene exons 1A and 4, respectively. Thus, the GHR gene is well conserved in the Bovidae family. Also, in this study some novel intraspecies polymorphisms were found for cattle and sheep.     

秦岭羚牛高度重复序列DNA片段的分子克隆和序列分析

羚牛(Budorcas taxicolor)属偶蹄目(Artiodactyla)、牛科(Bovidae),为我国一类大型珍贵保护动物。我们从其基因组中克隆得到若干约800bp的BamHI高度重复序列并对部分克隆进行了序列测定,发现它们显示了很高的同源性。利用其中一个单元为探针,对限制酶消化后的羚牛基因组DNA作杂交分析,发现其杂交谱带不具有个体及亚种间特异性,说明该重复序列在羚牛基因组中具有保守的分布和排列。在牛科动物中,羚牛BamHI片段与绵羊属和山羊属的相关序列具有高度同源性,而与水牛和家牛序列差异较大。这些结果为羚牛与羊亚科物种亲源关系较近的分类学观点提供了分子生物学证据。有证据表明,这些片段可能代表羚牛染色体着丝点的卫星DNA单体。     

Expression and localization of SWAP-70 in human fetomaternal interface and placenta during tubal pregnancy and normal placentation.

SWAP-70 has been demonstrated as a multiple functional signaling protein involved in formation of membrane ruffling induced by signal cascade of tyrosine kinase growth factor receptors. In the present study, the spatial and temporal expression pattern of SWAP-70 on human fetomaternal interface was investigated using specimens collected from tubal and normal pregnancies by in situ hybridization, immunohistochemistry, and Western blotting. Data showed an intense expression of SWAP-70 in trophoblasts at weeks 3-6 of fallopian implantation and at weeks 6-7 of normal pregnancy. The most intense expression was exhibited by those highly motile and invasive extravillous trophoblasts. From gestational week 8 on, the level of SWAP-70 in trophoblasts decreased significantly, and the signal was restricted in villous cytotrophoblast cells. In the in vitro cultured human trophoblast cell line, B6Tert-1, colocalization of SWAP-70 with F-actin was verified. Data in human placenta were similar to what we recently reported on rhesus monkey fetomaternal interface. Our results suggest that SWAP-70 may be involved in regulating migration and invasion of trophoblast cells during the processes of embryonic implantation and placentation in primates.     

秦岭羚牛高度重复序列DNA片段的分子克隆和序列分析

羚牛（Budorcas taxicolor)属偶蹄目（Artiodactyla)、牛科（Bovidae),为我国一类大型珍贵保护动物。我们从其基因组中克隆得到若干约800bp的BamHI高度重复序列并对部分克隆进行了序列测定,发现它们显示了很高的同源性。利用其中一个单元为探针,对限制酶消化后的羚牛基因组DNA作杂交分析,发现其杂交谱带不具有个体及亚种间特异性,说明该重复序列在羚牛基因组中具有保守的分布和排列。在牛科动物中,羚牛BamHI片段与绵羊属和山羊属的相关序列具有高度同源性,而与水牛和家牛序列差异较大。这些结果为羚牛与羊亚科物种亲源关系较近的分类学观点提供了分子生物学证据。有证据表明,这些片段可能代表羚牛染色体着丝点的卫星DNA单体。     

Phylogenetic analyses and improved resolution of the family Bovidae based on complete mitochondrial genomes

Efforts have been made to investigate the phylogeny of the family Bovidae; however, the relationships within this group still remain controversial. To further our understanding of the relationships, we sequenced the mitochondrial genome of the Himalayan goral, Naemorhedus goral, an IUCN Redlist near threatened conservation dependent species. Then we conducted molecular phylogenetic relationships of the Bovidae based on Bayesian and Maximum Likelihood methods. The results indicate that the basal divergence within the Bovidae is between the Bovinae and a strongly supported clade of the remaining Bovidae species. The two Neotragus species (the suni and pygmy antelope) clustered with the impala, Aepyceros melampus (Aepycerotinae), and together they formed the most basal of the non-Bovinae. All the genera of the Antilopinae clustered together except Neotragus, which suggested that the Antilopinae was a paraphyletic subfamily. The present study confirmed a close relationship between the genera Capricornis and Naemorhedus while supporting their designation as separate genera and suggested that the Capricornis-Naemorhedus-Ovibos clade (serows, gorals, and the muskox) should be placed in the Caprinae. Bison, Bos, and Tragelaphus (bison & cattle and kudus and nyalas) were paraphyletic. The very close relationship between Bison and Bos suggested that Bos and Bison should be integrated into a single Bos genus. Saiga and Pantholops (the Chiru or Tibetan Antelope), unique genera which have sometimes been lumped together, were placed in different groups: Saiga within the Antilopinae and Pantholops at the base of the Caprinae. Our results also supported a new taxonomy which places the three species of Hemitragus into three monospecific genera: the genus Hemitragus is restricted to the Himalayan tahr, and two new genera are created: Arabitragus for the Arabian tahr and Nilgiritragus for the Nilgiri tahr.     

Placental Syncytium Forms a Biophysical Barrier against Pathogen Invasion

Fetal syncytiotrophoblasts form a unique fused multinuclear surface that is bathed in maternal blood, and constitutes the main interface between fetus and mother. Syncytiotrophoblasts are exposed to pathogens circulating in maternal blood, and appear to have unique resistance mechanisms against microbial invasion. These are due in part to the lack of intercellular junctions and their receptors, the Achilles heel of polarized mononuclear epithelia. However, the syncytium is immune to receptor-independent invasion as well, suggesting additional general defense mechanisms against infection. The difficulty of maintaining and manipulating primary human syncytiotrophoblasts in culture makes it challenging to investigate the cellular and molecular basis of host defenses in this unique tissue. Here we present a novel system to study placental pathogenesis using murine trophoblast stem cells (mTSC) that can be differentiated into syncytiotrophoblasts and recapitulate human placental syncytium. Consistent with previous results in primary human organ cultures, murine syncytiotrophoblasts were found to be resistant to infection with Listeria monocytogenes via direct invasion and cell-to-cell spread. Atomic force microscopy of murine syncytiotrophoblasts demonstrated that these cells have a greater elastic modulus than mononuclear trophoblasts. Disruption of the unusually dense actin structure – a diffuse meshwork of microfilaments - with Cytochalasin D led to a decrease in its elastic modulus by 25%. This correlated with a small but significant increase in invasion of L. monocytogenes into murine and human syncytium. These results suggest that the syncytial actin cytoskeleton may form a general barrier against pathogen entry in humans and mice. Moreover, murine TSCs are a genetically tractable model system for the investigation of specific pathways in syncytial host defenses.     

Comparative histology of antelope placentomes

The histological structure of ruminant (family: Bovidae) placentomes in eight antelope species was compared to that of domestic cattle and sheep. The chorioallantoic villi differed in degree of branching, surface corrugation, and complexity of utero-placental junction. All species had the epitheliochorial type of placenta, with the epithelial lining of maternal caruncular crypts varying between cellular and syncytial types.     

Localization of hepatocyte growth factor activator inhibitor type 1 in Langhans' cells of human placenta

Activation of hepatocyte growth factor (HGF) is a crucial limiting step in HGF-induced signaling pathway. The HGF activator inhibitor type 1 (HAI-1) was identified as a potent inhibitor of HGF activator (HGFA), a serine proteinase that is responsible for the activation of HGF in vivo. HAI-1 is an integral membrane Kunitz-type serine proteinase inhibitor, and its mRNA has been reported to be most abundant in the placenta. In this report, specific antibody to HAI-1 was used in an immunohistochemical procedure to determine the localization of HAI-I in human placenta. HAI-1 was expressed in cytotrophoblasts (Langhans' cells) of the double-layered trophoblastic epithelium of chorionic villi tissue, and syncytiotrophoblasts were almost negative. On the other hand, extravillous trophoblasts of cytotrophoblastic columns showed markedly decreased immunoreactivity, and those infiltrating into the superficial decidua membrane of early placenta were hardly stainable. The amnionic epithelial cells were also immunostained intensely. The presence of HAI-1 mRNA was also confirmed in a cultured human cytotrophoblastic cell line. In addition to HAI-1, low but distinct expression of HGFA mRNA was observed in the placenta tissue and cultured cytotrophoblasts by using a sensitive RT-PCR method. Since HGF plays an essential role in the placenta development, expression of HAI-1 and HGFA may have an important regulatory role in the placenta. The localization of HAI-I in the proliferating trophoblastic stem cells (Langhans' cells), but not in syncytiotrophoblasts and extravillous trophoblasts, suggest a possible role of HAI-1 in the proliferation of trophoblasts.     

Differential expression of the metastasis suppressor KAI1 in decidual cells and trophoblast giant cells at the feto-maternal interface

Invasion of trophoblasts into maternal uterine tissue is essential for establishing mature feto-maternal circulation. The trophoblast invasion associated with placentation is similar to tumor invasion. In this study, we investigated the role of KAI1, an anti-metastasis factor, at the maternal-fetal interface during placentation. Mouse embryos were obtained from gestational days 5.5 (E5.5) to E13.5. Immunohistochemical analysis revealed that KAI1 was expressed on decidual cells around the track made when a fertilized ovum invaded the endometrium, at days E5.5 and E7.5, and on trophoblast giant cells, along the central maternal artery of the placenta at E9.5. KAI1 in trophoblast giant cells was increased at E11.5, and then decreased at E13.5. Furthermore, KAI1 was upregulated during the forskolinmediated trophoblastic differentiation of BeWo cells. Collectively, these results indicate that KAI1 is differentially expressed in decidual cells and trophoblasts at the maternal-fetal interface, suggesting that KAI1 prevents trophoblast invasion during placentation. [BMB Reports 2013; 46(10): 507-512]     

Cytogenetic aspects of phylogeny in the Bovidae. I. G-banding

An extensive G-banding study of karyotypes of 12 species of Bovidae has been undertaken in an attempt to trace homologies and patterns of evolution of karyotype phenotypes throughout the family. G-banding profiles revealed a considerable degree of chromosome-arm homology throughout the group, which also extended into the related superfamilies, the Giraffoidea and Cervoidea. The conservation of banding patterns in chromosome arms strongly indicates that Robertsonian translocation type rearrangements have provided the major source of interspecies karyotype differences, with inversions and reciprocal and tandem translocations providing relatively minor contributions. Examples of individuals carrying newly arisen Robertsonian translocations are not infrequent, and in one instance there was evidence that two similar rearrangements had arisen independently in two species. Despite the extensive changes in karyotype organization, subfamilies within the Bovidae were characterized by the presence of common rearrangements, and those involving autosomal pairs 11 and 12 of the ox, as well as the X chromosome, separate the Bovinae from the Caprinae and Hippotraginae.     

The tribal radiation of the family Bovidae (Artiodactyla) and the evolution of the mitochondrial cytochrome b gene

The nucleotide sequence of the complete mitochondrial cytochrome b gene has been determined and compared for 51 species of the family Bovidae and 10 potential pecoran and tragulid outgroups. A detailed saturation analysis at each codon position relative to the maximum parsimony procedure indicates that all transitions on third codon positions do not accumulate in a similar fashion: C-T are more saturated than A-G substitutions. The same trend is observed for second positions but not for first positions where A-G and C-T transitions exhibit roughly the same levels of saturation. Maximum parsimony reconstructions were weighted according to these observations. Maximum parsimony, maximum likelihood, and distance phylogenetic reconstructions all depict a major split within Bovidae. The subfamily Bovinae includes four multifurcating tribes and subtribes: Boselaphini, Tragelaphini, cattle-Bovini (Bos and Bison), and buffalo-Bovini (Bubalus and Syncerus). Its sister group is the subfamily Antilopinae, i.e., all non-Bovinae taxa, represented by seven lineages: Antilopini (including Saiga), Caprini sensu lato (i. e., Caprinae including Pantholops), Hippotragini, Alcelaphini, Reduncini (including Pelea), Aepyceros possibly linked to Neotragus, and Cephalophini possibly linked to Oreotragus (the suni and the klipspringer being members of a polyphyletic Neotragini). These various tribes and major lineages were produced by two noteworthy explosive radiations, which occurred simultaneously between 12.0 and 15.3 MY (Middle Miocene) in the subfamilies Bovinae and Antilopinae.     

Molecular insights into the evolution of the family Bovidae: a nuclear DNA perspective.

The evolutionary history of the family Bovidae remains controversial despite past comprehensive morphological and genetic investigations. In an effort to resolve some of the systematic uncertainties within the group, a combined molecular phylogeny was constructed based on four independent nuclear DNA markers (2,573 characters) and three mitochondrial DNA genes (1,690 characters) for 34 bovid taxa representing all seven of the currently recognized bovid subfamilies. The nuclear DNA fragments were analyzed separately and in combination after partition homogeneity tests were performed. There was no significant rate heterogeneity among lineages, and retention index values indicated the general absence of homoplasy in the nuclear DNA data. The conservative nuclear DNA data were remarkably effective in resolving associations among bovid subfamilies, which had a rapid radiation dating back to approximately 23 MYA. All analyses supported the monophyly of the Bovinae (cow, nilgai, and kudu clade) as a sister lineage to the remaining bovid subfamilies, and the data convincingly suggest that the subfamilies Alcelaphinae (hartebeest, tsessebe, and wildebeest group) and Hippotraginae (roan, sable, and gemsbok clade) share a close evolutionary relationship and together form a sister clade to the more primitive Caprinae (represented by sheep, goat, and muskox). The problematic Reduncinae (waterbuck, reedbuck) seem to be the earliest-diverging group of the Caprinae/Alcelaphinae/Hippotraginae clade, whereas the Antilopinae (gazelle and dwarf antelope clade) were always polyphyletic. The sequence data suggest that the initial diversification of the Bovidae took place in Eurasia and that lineages such as the Cephalophinae and other enigmatic taxa (impala, suni, and klipspringer) most likely originated, more or less contemporaneously, in Africa.     

Phylogenetics of the Caprinae Based on Cytochrome b Sequence

Relationships within the subfamily Caprinae have never been fully resolved. Phylogenies have been proposed based on morphological, behavioral, ecological, and some molecular comparisons. Because of the relatively poor fossil record of the Caprinae, paleontological evidence has not been extensively used in phylogenetic reconstruction for this group. Traditionally, four tribes: Saigini, Ovibovini, Rupicaprini, and Caprini, have been recognized. We investigated relationships within the Caprinae by comparing sequences of the cytochrome b gene of mitochondrial DNA from 11 species of Caprinae and 1 Bovinae species. Our analyses suggest that revisions to previous phylogenies, including the dissolution of the Ovibovini, are warranted.     

Role of the Vasohibin Family in the Regulation of Fetoplacental Vascularization and Syncytiotrophoblast Formation

Vasohibin-1 (VASH1) and vasohibin-2 (VASH2), the 2 members of the vasohibin family, have been identified as novel regulators of angiogenesis. VASH1 ceases angiogenesis, whereas VASH2 stimulates sprouting. Here we characterized their functional role in the placenta. Immunohistochemical analysis of human placental tissue clarified their distinctive localization; VASH1 in endothelial cells and VASH2 in trophoblasts. We then used a mouse model to explore their function. Wild-type, Vash1^(−/−), and Vash2^(−/−) mice on a C57BL6 background were used in their first pregnancy. As expected, the fetal vascular area was increased in the Vash1^(−/−) mice, whereas it was decreased in the Vash2^(−/−) mice relative to wild-type. In addition, we noticed that the Vash2^(−/−) mice at 18.5dpc displayed thinner villi of the labyrinth and larger maternal lacunae. Careful observation by an electron microscopy revealed that the syncytiotrophoblast formation was defective in the Vash2^(−/−) mice. To test the possible involvement of VASH2 in the syncytiotrophoblast formation, we examined the fusion of BeWo cells, a human trophoblastoid choriocarcinoma cell line. The forskolin treatment induced the fusion of BeWo cells, and the knockdown of VASH2 expression significantly inhibited this cell fusion. Conversely, the overexpression of VASH2 by the infection with adenovirus vector encoding human VASH2 gene significantly increased the fusion of BeWo cells. Glial cell missing-1 and endogenous retrovirus envelope glycoprotein Syncytin 1 and Syncytin 2 are known to be involved in the fusion of trophoblasts. However, VASH2 did not alter their expression in BeWo cells. These results indicate that VASH1 and VASH2 showed distinctive localization and opposing function on the fetoplacental vascularization. Moreover, our study shows for the first time that VASH2 expressed in trophoblasts is involved in the regulation of cell fusion for syncytiotrophoblast formation.