Sensitivity of expression of perivitelline membrane glycoprotein ZP1 mRNA in the liver of Japanese quail (Coturnix japonica) to estrogenic compounds

Avian perivitelline membrane protein, ZP1, is synthesized and secreted by the liver with the stimulation of estrogens. In the present study, we measured the expression of ZP1 gene in the liver of immature male quail treated with various estrogenic compounds and in the liver of male quail embryos that were developed in the fertilized eggs laid by mother quail injected with various estrogenic compounds during vitellogenesis. Total RNA extracted from the liver was reverse-transcribed and cDNA was subjected to real-time PCR. Both diethylstilbestrol and ethinyl estradiol caused significant effect on the increase in mRNA in immature male quail. In contrast, diethylstilbestrol administered via the route of maternal injection was not effective for induction of embryonic mRNA, although the effect of ethinyl estradiol administered via the same route was prominent. These results showed that direct administration of estrogenic compounds, diethylstilbestrol and ethinyl estradiol, stimulates the induction of ZP1 gene, but the rate of accumulation of these compounds in the yolk is different during vitellogenesis. The present studies suggest that although ZP1 gene is a sensitive biomarker to evaluate the effects of endocrine disruptors, the route of administration is an important factor to compare the effectiveness.     

Incorporation of ZP1 into perivitelline membrane after in vivo treatment with exogenous ZP1 in Japanese quail (Coturnix japonica)

In birds, the egg envelope surrounding the oocyte prior to ovulation is called the perivitelline membrane and it plays important roles in fertilization. In a previous study we demonstrated that one of the components of the perivitelline membrane, ZP3, which is secreted from the ovarian granulosa cells, specifically interacts with ZP1, another constituent that is synthesized in the liver of Japanese quail. In the present study, we investigated whether ZP1 injected exogenously into the blood possesses the ability to reconstruct the perivitelline membrane of Japanese quail. When ZP1 purified from the serum of laying quail was injected into other female birds, the signal of this exogenous ZP1 was detected in the perivitelline membrane. In addition, we revealed, by means of ligand blot analysis, that serum ZP1 interacts with both ZP1 and ZP3 of the perivitelline membrane. By contrast, when ZP1 derived from the perivitelline membrane was administered, it failed to become incorporated into the perivitelline membrane. Interestingly, serum ZP1 recovered from other Galliformes, including chicken and guinea fowl, could be incorporated into the quail perivitelline membrane, but the degree of interaction between quail ZP3 and ZP1 of the vitelline membrane of laid eggs from chicken and guinea fowl appeared to be weak. These results demonstrate that exogenous ZP1 purified from the serum, but not ZP1 from the perivitelline membrane, can become incorporated into the perivitelline membrane upon injection into other types of female birds. To our knowledge, this is the first demonstration that the egg envelope component, when exogenously administered to animals, can reconstruct the egg envelope in vivo.     

Species specificity in avian sperm:perivitelline interaction

The interaction of chicken spermatozoa with the inner perivitelline layer from different avian species in vitro during a 5 min co-incubation was measured as the number of points of hydrolysis produced per unit area of inner perivitelline layer. The average degree of interaction, as a proportion of that between chicken spermatozoa and their homologous inner perivitelline layer, was: equal to or greater than 100% within Galliformes (chicken, turkey, quail, pheasant, peafowl and guineafowl); 44% within Anseriformes (goose, duck); and less than 30% in Passeriformes (Zebra Finch) and Columbiformes (collared-dove). The homologue of the putative chicken sperm-binding proteins, chicken ZP1 and ZP3, were identified by Western blotting with anti-chicken ZP1/ZP3 antibody in the perivitelline layers of all species. The functional cross-reactivity between chicken spermatozoa and heterologous inner perivitelline layer appeared to be linked to known phylogenetic distance between the species, although it was not related to the relative affinity of the different ZP3 homologues for anti-chicken ZP3. This work demonstrates that sperm interaction with the egg investment does not represent such a stringent species-specific barrier in birds as it does in mammals and marine invertebrates. This may be a factor in the frequency of hybrid production in birds.     

Cloning and characterization of a novel ITIM containing lectin-like immunoreceptor LLIR and its two transmembrane region deletion variants

A novel full-length cDNA was cloned from human dendritic cells (DC) by subtractive cloning and RACE. The deduced protein is a type II lectin-like membrane protein that contains an ITIM proximal to N terminal and is designated as lectin-like immunoreceptor (LLIR). The gene of LLIR is located in a region of chromosomal 12p13 and shows highest homologous with ASGPR. Two alternatively spliced transmembraneless variants of LLIR were identified by RT-PCR and named as LLIRv1 and LLIRv2. RT-PCR and immunoblotting analysis revealed that LLIR was expressed with much higher level in immature DC than in mature DC. The ITIM in LLIR was demonstrated to bind SHP-1 in HL-60 cell after the tyrosine had been phosphorylated. In addition, the mRNA expression level of LLIRv2 was raised when leukemia cells were induced to differentiate by PMA.     

Isolation and characterization of a full-length cDNA encoding the 55-kDa rabbit zona pellucida protein.

A full-length cDNA (rc55) encoding the major rabbit zona pellucida (ZP) glycoprotein (55 kDa) has been cloned and sequenced. A lambda gt11 expression library was constructed using poly(A)+ mRNA isolated from sexually immature rabbit ovaries which contain large numbers of developing follicles. The rc55 cDNA was identified using affinity purified polyclonal antibodies specific to ZP antigens which are shared among mammalian species. The deduced amino acid sequence of the full-length rc55 clone was matched to the NH2-terminal 25-amino acid sequence obtained for this protein. The predicted amino acid sequence consists of 540 amino acids including a putative signal peptide of 18-24 residues and six potential N-glycosylation sites. The cDNA hybridizes to a 2000-base species of mRNA from rabbit ovary which is not detected in other rabbit tissues. The message is present early in ovarian follicular development and is approximately 600-fold greater in sexually immature as compared with sexually mature rabbit ovaries. This cDNA was expressed as a cro-beta-galactosidase fusion protein using the pEX expression vector. Antibodies against native rabbit ZP, affinity-purified on the recombinant 55-kDa ZP protein, were found to recognize the native rabbit ZP glycoprotein, indicating partial conservation of native epitopes in the expressed recombinant protein.     

人ZP3基因的RT-PCR cDNA克隆

目的：研究人ZP3基因的结构及构建人ZP3基因原核表达系统。方法：从人卵巢组织中分离出mRNA并以此作为模版,通过RT—PCR扩增出人ZP3基因cDNA片段,然后将其克隆在pUC18质粒上,并对克隆片段进行序列分析。结果：共克隆到ZP3-A（1300bp）、ZP3-B（1180bp）、ZP3-C（1200bp）和ZP3-D（1080bp）4种不同长度的人ZP3基因cDNA片段,对其中最长的ZP3-A片段的测序结果表明,它包含了人ZP3基因阅读框内的全部序列,与NCBI Sequence Viewer中公布的人ZP3 mRNA序列（NM-007155）相比较,在1275bp长的编码区内只有一个碱基不同,两者同源性达到99．92％。结论：本研究克隆到的ZP3-A cDNA片段确是人ZP3基因无疑。     

Schistosoma mansoni: SmLIMPETin, a member of a novel family of invertebrate-only regulatory proteins

Eukaryotic LIM domain proteins contain zinc finger forming motifs rich in cysteine and histidine that enable them to interact with other proteins. A cDNA clone isolated from an adult schistosome cDNA library revealed a sequence that coded for a novel class of proteins bearing 6 LIM domains and an N-terminal PET domain, SmLIMPETin. Phylogeny reconstruction of SmLIMPETin and comparison of its sequence to invertebrate homologues and to the vertebrate four-and-a-half LIM domains protein family (FHLs), uncovered a novel LIM domain protein family, the invertebrate LIM and PET domain protein family (LIMPETin). Northern blots, RT-PCR and Western blot showed that SmLIMPETin gene was less expressed in sexually mature adult females compared to sexually immature adult females and sexually mature and immature adult males, and not expressed in schistosomula.     

Growth differences and dimorphic expression of growth hormone (GH) in female and male Cynoglossus semilaevis after male sexual maturation

Half-smooth tongue sole, Cynoglossus semilaevis, is an ideal model to investigate the regulatory mechanisms of sexual growth dimorphism in fish species. The aim of the study was to investigate the effect of differential age of sexual maturity for females and males on growth and GH mRNA expression in C. semilaevis. The body weight differences between the sexes were not significant in C. semilaevis at age 5 months when females and males were all immature. Significant differences in body weight between the sexes were found after early sexual maturation of males at the age of 9 months. The body weight of 21-month-old females (621.4 ± 86.4g), still not immature, was even 3.28 times higher than that of the males (189.7 ± 14.4g). The cDNAs encoding GH in C. semilaevis was cloned. The GH gene is 2924bp long and consists of six exons and five introns. The results of qRT-PCR showed that GH mRNA levels of the immature females were not significantly different from that of immature males at age 5 months. However, GH mRNA levels of the immature females were significantly higher compared with those of the mature males at age 9 months (P<0.05). At age 11 months, GH mRNA levels of females were even 6.4-fold higher than that of males. In conclusion, for the first time we show that early sexual maturity of males is the main cause of sexual growth dimorphism in C. semilaevis and exert significant effect on GH mRNA expression.     

Isolation and characterization of a cDNA encoding a serine proteinase from the root-knot nematode Meloidogyne incognita

This report describes the first serine proteinase gene isolated from the sedentary nematode Meloidogyne incognita. Using degenerate primers, a 1372bp cDNA encoding a chymotrypsin-like serine proteinase (Mi-ser1) was amplified from total RNA of adult females by RT-PCR and 5' and 3' rapid amplification of cDNA ends. The deduced amino acid sequence of Mi-ser1 encoded a putative signal peptide and a prodomain of 22 and 33 amino acids, respectively, and a mature proteinase of 341 amino acids with a predicted molecular mass of 37,680Da. Sequence identity with the top serine proteinases matches from the databases ranged from 23 to 27%, including sequences from insects, mammals, and other nematodes. Southern blot analysis suggested that Mi-ser1 is encoded by a single or few gene copies. The pattern of developmental expression analyzed by Northern blot and RT-PCR indicated that Mi-ser1 was transcribed mainly in females. The domain architecture composed of a single chymotrypsin-like catalytic domain and the detection of a putative signal peptide suggested a digestive role for Mi-ser1.     

Gene expression in mouse oocytes and preimplantation embryos: use of suppression subtractive hybridization to identify oocyte- and embryo-specific genes

The paucity of biological material has inhibited identifying genes that are differentially expressed during mammalian oogenesis and preimplantation development. We report here the linear amplification of mRNA from small numbers of mouse oocytes and preimplantation embryos to generate amounts of sense RNA that are sufficient for suppression subtractive hybridization. The resulting oocyte-specific and 8-cell-specific cDNA libraries were partially characterized, and the known oocyte-specific ZP1, ZP2, GDF-9, BMP15, and H1(oo) genes were found in the oocyte-specific cDNA library but not in the 8-cell-specific library. Further characterization of the subtracted oocyte and 8-cell embryo cDNA libraries should furnish a trove of information regarding temporal changes in gene expression during oogenesis and preimplantation development in the mouse.     

Involvement of interaction of ZP1 and ZPC in the formation of quail perivitelline membrane

The extracellular matrix surrounding the oocyte before ovulation is called the perivitelline membrane (PL) in avian species. We have previously reported that one of its components, ZPC, is produced in ovarian granulosa cells by the stimulation of follicle-stimulating hormone and testosterone. Another component, ZP1, is synthesized in the liver and might be transported to the surface of the oocyte of the follicles. These glycoproteins are assembled to form a three-dimensional network of coarse fibers between the granulosa cells and the oocyte. In the present study, we have evaluated the involvement of the interaction of ZPC and ZP1 in the formation of the PL of Japanese quail. By measuring the incorporation of tritium-labeled proteins into the PL, we have found that tritium-labeled ZPC is specifically incorporated into the PL. Whole-mount autoradiographic analysis of the PL has also revealed the incorporation of the secreted ZPC into the isolated PL. To study which component in the PL is responsible for the specific incorporation of ZPC, PL lysates were incubated with the conditioned medium of the granulosa cells and were immunoprecipitated with anti-ZPC antiserum. Western blot analysis of the immunoprecipitated materials indicated that the 175-kDa and 97-kDa ZP1 forms were co-immunoprecipitated with anti-ZPC antiserum. These results demonstrate that ZPC secreted from the granulosa cells specifically binds with ZP1, and that the phenomenon might be involved in insoluble PL fiber formation in quail ovary.Funding for this work was provided by the Ministry of Education, Science, Sports and Culture of Japan (Grant-in-Aids for Scientific Research: 14760177 and 16780192 to T.S. and 15380191 to M.M.)     

Molecular characterization of a cDNA encoding vitellogenin in the banana shrimp, Penaeus (Litopenaeus) merguiensis and sites of vitellogenin mRNA expression

In order to determine the primary structure of banana shrimp, Penaeus merguiensis, vitellogenin (Vg), we previously purified vitellin (Vt) from the ovaries of vitellogenic females, and chemically analyzed the N-terminal amino acid sequence of its 78 kDa subunit. In this study, a cDNA from this species encoding Vg was cloned based on the N-terminal amino acid sequence of the major 78 kDa subunit of Vt and conserved sequences of Vg/Vt from other crustacean species. The complete nucleotide sequence of Vg cDNA was achieved by RT-PCR and 5' and 3' rapid amplification of cDNA ends (RACE) approaches. The full-length Vg cDNA consisted of 7,961 nucleotides. The open reading frame of this cDNA encoding a precursor peptide was comprised of 2,586 amino acid residues, with a putative processing site, R-X-K/R-R, recognized by subtilisin-like endoproteases. The deduced amino acid sequence was obtained from the Vg cDNA and its amino acid composition showed a high similarity to that of purified Vt. The deduced primary structure, of P. merguiensis Vg was 91.4% identical to the Vg of Penaeus semisulcatus and was also related to the Vg sequences of six other crustacean species with identities that ranged from 86.9% to 36.6%. In addition, the amino acid sequences corresponding to the signal peptide, N-terminal region and C-terminal region of P. merguiensis Vg were almost identical to the same sequences of the seven other reported crustacean species. Results from RT-PCR analysis showed that Vg mRNA expression was present in both the ovary and hepatopancreas of vitellogenic females but was not detected in other tissues including muscle, heart, and intestine of females or in the hepatopancreas of mature males. These results indicate that the Vg gene may be expressed only by mature P. merguiensis females and that both the ovary and hepatopancreas are possible sites for Vg synthesis in this species of shrimp.     

Molecular characterization and expression profile of a novel porcine gene differentially expressed in the muscle and backfat tissues from Chinese Meishan and Russian Large White pigs

The mRNA differential display technique was performed to investigate the differences of gene expression in the longissimus dorsi muscle and backfat tissues from Chinese Meishan and Russian Large White pigs. One novel gene that was differentially expressed was identified through semi-quantitative RT-PCR and the cDNA complete sequence was then obtained using the rapid amplification of cDNA ends (RACE) method. The cDNA sequence of this gene is not homologous to any of the known porcine genes. The sequence prediction analysis revealed that the open reading frame of this gene encodes a protein of 402 amino acids that contains the putative conserved transposase DDE domain and further Blast analysis revealed that this protein has 100% homology with the Tn10 transposase from Oryza sativa, Serratia marcescens, and Salmonella, and therefore, this gene can be defined as the swine Tn10 transposase gene. This novel porcine gene was finally assigned to Gene ID: 100049649. The RT-PCR analysis of the tissue expression profile was carried out using the tissue cDNAs of one Meishan pig as the templates, and the result indicated that this novel swine gene is moderately expressed in fat, and weakly expressed in small intestine, liver, kidney, and spleen but almost not expressed in heart, ovary, muscle, and lung. Our experiment established the primary foundation for further research into the biological significance of swine Tn10 transposase gene.     

Luteinizing hormone secretion from the quail pituitary in vitro

An enzymatically dispersed pituitary preparation from Japanese quail (Coturnix coturnix) was used to study the dynamics of gonadotropin release. After an 18-h incubation, the cells were challenged with different luteinizing hormone-releasing hormones (LHRH) for 90 min. Using pituitary cells from mature males, mammalian and chicken LHRH I (Gln8-LHRH) had approximately equal luteinizing hormone (LH)-releasing activity whereas chicken LHRH II (His5, Trp7, Tyr8-LHRH) was 8-9 times more potent. The LHRH agonist (Trp6, Pro9-NEt-LHRH) had 15 times greater potency than chicken LHRH I. Pre-incubation with an LHRH antagonist (D-Phe2, D-Trp6-LHRH) significantly suppressed LH release. Acid extracts of median eminence released LH from pituitary cells, extracts from short-day and long-day males had equal activity, while tissue extracts from castrated males had significantly greater LH-releasing activity. Pituitary cells from sexually immature males released LH in response to chicken LHRH I in a similar profile to cells from mature males. These data indicate that the quail LHRH receptor in the male recognizes several different molecular species of LHRH and the response to LHRH is comparable between short- and long-day males. Pituitary cells from ovulating females were variably sensitive to LHRH peptides, possibly due to changes in pituitary sensitivity during the ovulatory cycle. Pituitary cells from immature females did not release LH in response to chicken LHRH I. However, pituitary cells from immature females photostimulated for 1 wk displayed a response to chicken LHRH I and II similar to that of pituitary cells from males.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genomic structure, polymorphism and expression analysis of the growth hormone (GH) gene in female and male Half-smooth tongue sole (Cynoglossus semilaevis)

Growth hormone (GH) is a polypeptide which is an important regulator of development and somatic growth in teleosts, and may be associated with the mechanisms which drive sexual growth dimorphism in the Half-smooth tongue sole (Cynoglossus semilaevis). In this study, the full length gh cDNA was cloned from C. semilaevis by homology cloning and the rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR). The full-length gh cDNA is 826 bp and contains an open reading frame (ORF) of 603 bp encoding a protein of 200 amino acids (AA). The precursor of gh consists of a 17 amino-acid signal peptide followed by a 183 amino-acid mature polypeptide. GH gene sequences obtained from female and male adults consist of 3428 bp and 3371 bp, respectively, each of which includes six exons and five introns, and the difference in the GH gene size was mainly caused by the microsatellites. When 14 tissues from females, normal males and extra-large male adults were analyzed for sex-specific tissue expression, the gh mRNA was found to be predominantly expressed in the pituitary, and the expression levels in females were 3.6 times as much as those in normal males, while the mRNA expression in extra-large males was 1.7 times as much as those in normal males. Sex differences in gh mRNA expression during development were also examined by using a full-sib family of C. semilaevis, and the gh mRNA was detected at all of the 12 time points sampled from 10 to 380 days-old. A significant increase in gh mRNA was detected starting in 80 day old fish and was then followed by a drop to very low levels starting at 230 day old fish. Differential expression indicated that the gh expression level in females was significantly higher than males (P < 0.01) at all of the stages except for 10 days-old. Two microsatellite loci were identified in the second intron of the GH gene. Using these two polymorphic markers to genotype 224 individuals, there was no significant difference between the females and males in the Bohai Sea, the Yellow Sea and the hatchery samples.     

Molecular characterization and expression profile of a novel porcine gene differentially expressed in the muscle and backfat tissues from Chinese Meishan and Russian Large White pigs

The mRNA differential display technique was performed to investigate the differences of gene expression in the longissimus dorsi muscle and backfat tissues from Chinese Meishan and Russian Large White pigs. One novel gene that was differentially expressed was identified through semiquantitative RT-PCR, and the cDNA complete sequence was then obtained using the rapid amplification of the cDNA ends (RACE) method. The cDNA sequence of this gene is not homologous to any of the known porcine genes. The sequence prediction analysis revealed that the open reading frame of this gene encodes a protein of 402 amino acids that contains the putative conserved transposase DDE domain, and further Blast analysis revealed that this protein has 100% homology with the Tn10 transposase from Oryza sativa, Serratia marcescens, and Salmonella, and, therefore, this gene can be defined as the swine Tn10 transposase gene. This novel porcine gene was finally assigned to Gene ID: 100049649. The RT-PCR analysis of the tissue expression profile was carried out using the tissue cDNAs of one Meishan pig as the templates, and the result indicated that this novel swine gene is moderately expressed in fat and weakly expressed in small intestine, liver, kidney, and spleen but almost not expressed in heart, ovary, muscle, and lung. Our experiment established the primary foundation for further research into the biological significance of swine Tn10 transposase gene.