The putative promoters of germ cell-specific genes and Nanog are hypomethylated in chicken sperm.

Germ cell-specific genes such as Ddx4, Dnd1, and Dazl play critical roles in the proliferation and survival of germ cells. However, the methylation state of the promoter in mature germ cells is still unknown. Here, we investigated the methylation levels of these genes and the pluripotency marker gene Nanog in chicken sperm as compared with the Alb gene in the liver. CpG islands and/or promoter motifs such as TATA box, GC box and CAAT box were found within the putative promoter regions that we identified. By using the bisulfite reaction, CpG sites in the putative promoters were converted, and they were analyzed by sequencing. The putative promoters of Ddx4, Dnd1, Dazl and Nanog showed very low methylation levels in sperm, but they were highly methylated in the liver. Conversely, the Alb gene promoter was highly methylated in sperm and hypomethylated in the liver. However, no transcripts of Ddx4, Dnd1, Dazl and Nanog were detected in sperm or the liver. Also, no transcripts of Dnmt1 and Dnmt3a were detected in sperm. Our present results may indicate that these germ cell-specific genes and the pluripotency marker gene are ready to express any time after fertilization. Our findings showing that low methylation and selective DNA methylation of specific genes are present in chicken sperm contribute to our understanding of fertilization and embryogenesis of birds.

Development of a wireless sensor for the measurement of chicken blood flow using the laser Doppler blood flow meter technique.

Here, we report the development of an integrated laser Doppler blood flow micrometer for chickens. This sensor weighs only 18 g and is one of the smallest-sized blood flow meters, with no wired line, these are features necessary for attaching the sensor to the chicken. The structure of the sensor chip consists of two silicon cavities with a photo diode and a laser diode, which was achieved using the microelectromechanical systems technique, resulting in its small size and significantly low power consumption. In addition, we introduced an intermittent measuring arrangement in the measuring system to reduce power consumption and to enable the sensor to work longer. We were successfully able to measure chicken blood flow for five consecutive days, and discovered that chicken blood flow shows daily fluctuations.

Colocalization of nitric oxide synthase, vasoactive intestinal polypeptide and tyrosine hydroxylase immunoreactivities in postganglionic neurons of the quail superior cervical ganglion.

The colocalization of immunoreactivity to nitric oxide synthase (NOS), vasoactive intestinal polypeptide (VIP) and tyrosine hydroxylase (TH) in the superior cervical ganglion (SCG) was investigated in the quail. In this bird, a substantial amount of NOS-immunoreactive (IR) cells were consistently found in the SCG without colchicine treatment or nerve ligation. The finding worthy of pointing out was that three-fourths of these NOS-IR cells were positive for TH. VIP-IR cells appeared with markedly low frequency than NOS-IR cells. They were generally small in size and often located in the ganglion peripheral. There were no VIP-IR cells positive for TH or negative for NOS: VIP immunoreactivity always appears in NOS-IR cells negative for TH. Thus, the results of the present study clearly showed the existence of two distinct subpopulations of postganglionic NOS-IR neurons (one is catecholaminergic and negative for VIP, and the other is non-catecholaminergic and positive for VIP). This suggests that nitric oxide (NO) and possibly VIP act as postganglionic neurotransmitters or neuromodulators in the quail SCG. The predominant appearance of the former category of NOS-IR cells must be considered in relation to some specific NO-induced controlling mechanisms of SCG neurons.

Steroidal regulation of Ihh and Gli1 expression in the rat uterus.

Ovarian steroid hormones, progesterone (P4), and estradiol (E2) strictly regulate the endometrial tissue remodeling required for successful embryo implantation. Indian hedgehog (Ihh) is up-regulated by P4 and critically mediates uterine receptivity in the mouse. However, the regulation of Ihh expression during the implantation period still remains unclear. The present study was conducted to elucidate the mechanism of the steroidal regulation in the expression of Ihh and Gli1, the mediator of the Ihh pathway. Ihh mRNA was expressed in the rat uterus on 3.5-5.5 days post-coitus (dpc), while Gli1 expression transiently increased at 3.5 dpc but decreased significantly on 5.5 dpc (P < 0.001). In delayed implantation, the expression of Ihh was induced by the implantation-induced E2 treatment in the primed rat uterus. In contrast, expression of Gli1 was significantly decreased by E2 treatment (P = 0.016). In the case of ICI182.780 (ICI) treatment, Ihh expression was eliminated by ICI, whilst Gli1 expression increased. These results suggest that Ihh expression is maintained at a high level until the initiation of implantation, while the expression of Gli1 is decreased just prior to the initiation of implantation depending on the E2 action. This observation aids in the understanding of the Ihh signaling pathway mediating uterine remodeling for implantation.

Temporal and spatial differential expression of chicken germline-specific proteins cDAZL, CDH and CVH during gametogenesis.

The Deleted in Azoospermia-Like (DAZL) protein coded by Dazl gene is a germline-specific RNA-binding protein essential for gametogenesis in vertebrates, and the chicken Dazl gene has also been identified in primordial germ cells (PGCs). However, the temporal and spatial expression of chicken DAZL (cDAZL) and its molecular role in germ cell development remain enigmatic. Here, we investigated the subcellular distribution and expression of cDAZL at the various stages by using a polyclonal antibody raised against its C-terminal region and compared them with those of additional germline-specific proteins chicken vasa homologue (CVH) and chicken dead end homologue (CDH). Western blot analysis for cDAZL revealed a single band in the embryonic gonads and premature chicken testis, whereas no band was detected in the premature chicken ovary. Fluorescent immunohistochemistry revealed that cDAZL was present in the nucleus and cytoplasm of circulating PGCs. Cells positive for cDAZL and CVH coexisted in the embryonic gonads and premature chicken testis, in which they were distributed near the basement membrane of seminiferous tubules. Of interest, cDAZL was not found in the premature chicken ovary, whereas CVH and CDH were present in germ cells. Collectively, three germline-specific proteins are expressed in chicken germ cells, but their patterns of expression are temporally and spatially distinct.

Monitor of the myostatin autocrine action during differentiation of embryonic chicken myoblasts into myotubes: effect of IGF-I.

Myogenesis is regulated through the proliferation and differentiation of myoblasts expressing myostatin which functions as a negative regulator by generating Smad signals. Here, we monitored the autocrine action of myostatin in quiescent chicken myoblasts transfected with the Smad-mediated promoter reporter vector to evaluate the modulation of several growth factors. During differentiation of myoblasts into myotubes, stretched and spherical types of myoblasts were observed at 12 h after induction, at which the promoter activity began to increase. Maximal promoter activity was observed at approximately 30 h. Multinucleated myotubes were markedly formed at 72 h, but the activity was very low. IGF-I, known as a positive regulator of myogenesis, increased the promoter activity, but the increase was rather small at its high concentration (100 ng/ml). IGF-I significantly increased the level of myostatin transcript in myoblasts and newly formed myotubes at 24 h, but not at 36 h. However, the cell fusion of myoblasts was not accelerated in the presence of IGF-I. Consequently, this study indicates that the autocrine action of myostatin is partially enhanced by IGF-I through increasing its expression.

Chicken dead end homologue protein is a nucleoprotein of germ cells including primordial germ cells.

The dead end gene, coding an RNA binding protein, is predominantly expressed in the germ cells of vertebrates. Recently, we cloned chicken dead end homologue (CDH) and showed that expression of CDH mRNA is highly specific to primordial germ cells (PGCs) at early embryonic stages. To date, the subcelluler localization of Dead end protein in germ cell has been largely unknown due to lack of an antibody. Here, we raised a polyclonal antibody against chicken dead end homologue (CDH) to elucidate its subcellular localization in the germ cells. For comparative studies with CDH, a polyclonal antibody against chicken vasa homologue (CVH), a well-known germ cell marker, was also raised. Immunoblotting analysis for CDH protein showed a single band with a molecular size of approximately 60 kDa in the ovarian and testicular proteins. Immunofluorescence studies revealed that CDH protein was exclusively localized in the nuclei of primordial germ cells (PGCs) and germ cells at later stages, while CVH was localized in the cytoplasm. Interestingly, the germ cells distributed at the basal sides of seminiferous epithelia, such as spermatogonia, were strongly positive to CDH protein. The current study provides novel evidence that CDH is a nucleoprotein of germ cells, including PGCs.

Development of vasoactive intestinal polypeptide-immunoreactive nerves in the major cerebral arteries of the quail anterior circulation.

Development of cerebral perivascular nerves immunoreactive for vasoactive intestinal polypeptide (VIP) was investigated in the Japanese quails, using immunohistochemistry and quantitative analysis. VIP-immunoreactive (VIP-IR) nerves supplying the anterior circulation appeared on the cerebral carotid artery (CCA) at embryonic day 10 and on the cerebroethmoidal artery (CEA) after hatching. Nerves from the CCA increased greatly in number and spread progressively during successive embryonic stages, while those from the CEA were sparse all through the post-hatching stages, mostly remained limited to this vessel wall. The distribution of VIP-IR nerves to the respective major arteries of the anterior circulation from the two vascular routes was basically similar among post-hatching day (P) 15, P20, P30 and P50. Likewise, no clear statistical difference was observed with regard to the nerve density of the corresponding arteries in the four age groups. These findings suggest that VIP-IR innervation of the quail anterior circulation usually attains its mature pattern at the third week after hatching.

In vitro decidualization of rat endometrial stromal cells.

The induction of the decidualization of endometrial stromal cells is possible in an in vitro cell culture system. However, thus far, methods differ according to species or cell type, and a more stable or universal system has not yet been developed. The purpose of the present study has been to establish an in vitro decidualization system in primary cultured rat endometrial stromal cells (RES). The RES were treated with medroxyprogesterone acetate and dibutyryl-cyclic adenosine monophosphate (MPA treatment), estradiol and progesterone, or arachidonic acid. After 24 h of treatment, cells responded to all of the stimulations by expressing desmin mRNA. However, decidual/trophoblast prolactin-related protein (dPRP) mRNA was only expressed in the MPA-treated cells. Desmin and dPRP mRNA were not expressed after MPA treatment of the RES derived from immature rat uteri. However, mRNA from both desmin and dPRP were expressed in RES derived from gonadotrophin-injected immature rats. The expression of matrix metalloproteinase-2 (MMP-2) and MMP-9 mRNA did not change after the decidual treatment of RES examined by real-time polymerase chain reaction. However, the results of gelatin zymography showed that the active forms of MMP-2 and MMP-9 significantly increased after in vitro decidualization (P < 0.05). We conclude that MPA treatment is the most effective method for stimulating decidualization in RES. Use of this system has revealed that sexual maturation and gonadotrophins are important for RES with regard to decidualization. Furthermore, the activity of MMP-2 and MMP-9 might increase during decidualization without a corresponding increase of the expression of these genes.

Expression of rat uterine serine proteinases homologous to mouse implantation serine proteinase 2.

Implantation serine protease (ISP) was first identified in the uteri of pregnant mice. It is thought that ISP may have an important role in the initiation of implantation. However, the expression status and detailed functions of ISP remain unclear. In this study, the expression of ISP was investigated in the rat uterus. The analysis of two rat genes registered in GenBank, accession nos. XM_220240 and XM_577076, exhibited high identities to the mouse ISP2 genes, respectively at an mRNA level. We labeled the former as rISP2a and the latter as rISP2b. Using RT-PCR, we found that both genes were expressed in the uterus. Specifically, rISP2a mRNA was detected in the uterus throughout pregnancy, whereas rISP2b mRNA was only expressed in the uterus from day 5 of pregnancy until the end of gestation. Expression of both genes was observed specifically within the endometrial gland epithelium. Furthermore, rISP2a was also observed to be expressed in the fetus and placenta, whereas rISP2b expression was observed in the fetus but not in the placenta. An expressional signal of the rISP2a gene was observed in the spongiotrophoblasts, giant cells and decidual endometrium in the placenta. In the embryo, the ventral specific region was positive in rISP2a and rISP2b gene expression. These findings indicate the possibility that the presently examined genes with high identity to mouse ISP2 may play some role not only during the implantation phase, but also in the development of the placenta and embryo.

Expression of hedgehog family genes in the rat uterus during early pregnancy.

Hedgehog (Hh) plays a pivotal role in various tissues during embryonic development, tissue homeostasis and tumorigenesis. In mammals, Hh exists in three homologs: Desert hedgehog (Dhh), Indian hedgehog (Ihh) and Sonic hedgehog (Shh). In this study, we cloned full-length cDNAs encoding Dhh and Ihh from the rat uterus. Their amino acid sequences have a high homology with those of the mouse and human. In addition, the changes of Hh gene expression in the rat uterus during early pregnancy were analyzed. The results showed that all three hedgehog mRNAs were detected in the rat uterus at the proestrus stage and during early pregnancy (1.5, 3.5, 5.5 and 7.5 days post coitus: dpc). Ihh mRNA expression varied and peaked at 3.5 dpc in the luminal and glandular epithelium. Expression was decreased on 5.5 dpc with the exception of sustained expression in the glandular epithelium. Despite such Ihh variability, the expressions of Dhh and Shh mRNA remained unchanged. This indicated that Ihh was mainly expressed in the rat uterus during early pregnancy. Moreover, the Hh target gene (glioma-associated oncogene homolog 1; Gli1) was also highly expressed at 3.5 dpc in the epithelium and periepithelial stroma in a manner similar to the temporal pattern of Ihh expression. This suggests that Ihh signaling axis play a role in the rat uterus during early pregnancy. In summary, our results elucidate that Ihh is a predominant Hh protein in the rat uterus during early pregnancy and that other Hhs have the potential to be expressed. This observation will help to elucidate the basic molecular mechanism of rat uterus during early pregnancy.

Effects of tumor necrosis factor-alpha on cell proliferation, prostaglandins and matrix-metalloproteinases production in rat endometrial stromal cells cultured in vitro.

Tumor necrosis factor-alpha (TNF-alpha) is known as a pluripotent cell mediator, and it is implicated in the control of uterine cell growth, differentiation and function during estrous cycle and pregnancy. In this study, we investigated the effect of TNF-alpha on endometrial stromal cells derived from rat uterus (rat endometrial stromal cells, RES). RES were isolated from rat endometrium at day 5 of pregnancy. Proliferation activities of RES were measured by using bromodeoxyuridine (BrdU) labeling kit, the productions of prostaglandin E2 (PGE2) and prostaglandin F2alpha (PGF2alpha) were measured by enzyme immunoassay kits and the production of matrix metalloproteinases (MMPs) was analyzed by gelatin-zymography. TNF-alpha, as well as epidermal growth factor and fibroblast growth factor-2, significantly increased the proliferation activity of RES (P<0.05). TNF-alpha selectively stimulated the production of PGE2 in RES (P<0.05), but not the production of PGF2alpha. Additionally, TNF-alpha did not stimulate the production of MMPs in RES at the concentration of 5 ng/mL, compared with the control groups (P>0.05). In conclusion, this study demonstrates several regulational functions of TNF-alpha on RES using in vitro culture system. The effects of TNF-alpha on proliferation and MMP production of RES have been shown for the first time. We believe that these results demonstrate part of the functions of TNF-alpha in endometrium and contribute to the better understanding of endometrial functions.

Ubiquitous expression of myostatin in chicken embryonic tissues: its high expression in testis and ovary.

The skeletal muscle of mammals is known to express myostatin (GDF-8) that acts as a potent negative regulator of skeletal muscle growth. However, the function of GDF-8 is not limited to skeletal muscle, because of its ubiquitous expression in fish. Here we investigated whether GDF-8 is expressed in various tissues including gonads during chicken embryogenesis. As revealed by RT-PCR and Western blotting, the transcript and protein for GDF-8 were detected in brain, eye, gizzard, muscle, heart, small gut, large gut, mesonephroi, testis and ovary of chicken embryos at E12, but not in liver. GDF-8 was constitutively expressed in testis and ovary as well as muscle at E6-E21, as demonstrated by in situ hybridization on section and whole-mount. Some cell population in testis, but not identified, highly expressed GDF-8. On the other hand, the medulla and germinal epithelium of ovary highly expressed it. Collectively, these results indicate that GDF-8 is ubiquitously expressed in various tissues of chicken embryos including testis and ovary through the stage of embryogenesis.

Molecular cloning and expression of dead end homologue in chicken primordial germ cells.

Chicken primordial germ cells (PGCs) dynamically migrate towards the prospective gonadal area through the germinal crescent region and bloodstream at early embryonic stages. To date, chicken PGCs have been mainly identified by histochemical and immunohistochemical methods or by their morphological characteristics. However, their origin, migration and differentiation are not fully understood because of the lack of specific PGC molecular markers. Here, we have isolated the chicken dead end homologue (CDH) in order to clone its full-length cDNA with an open reading frame of 329 amino acids. The RNA-binding motif present in CDH at amino acids 54-133 was highly homologous to those in the dead end proteins of human, mouse and Xenopus. The temporal and spatial distribution of PGCs was also investigated by in situ hybridization (ISH) on whole-mount embryos with CDH cRNA as a probe. Chicken embryos from stage X to stage 20 were subjected to ISH. The hybridized samples were then sectioned to analyse the translocation of PGCs. CDH-positive cells could be counted from stage X to stage 4, with minimally 30 cells at the blastderm and approximately 260 cells at the germinal crescent. Thus, specific expression of CDH mRNA has been established in chicken PGCs located at the blastderm, germinal crescent and prospective gonadal area by ISH and reverse transcription/polymerase chain reaction. We conclude that isolated CDH is specifically expressed in chicken PGCs during embryogenesis.

Different patterns of vasoactive intestinal polypeptide (VIP)-immunoreactive and acetylcholinesterase (AChE)-positive innervation in the internal carotid artery and cerebral arterial tree of the quail.

The innervation pattern of vasoactive intestinal polypeptide-immunoreactive (VIP-IR) nerves in the quail internal carotid artery (ICA) and cerebral arterial tree was investigated and compared with that of acetylcholinesterase-positive (AChE-P) nerves. The supply of VIP-IR nerves to the two arterial systems was distinctly richer than that of AChE-P nerves. It was focused mainly on the walls from the distal ICA to the caudal half of the anterior ramus (AR) through the cerebral carotid artery (CCA). Indeed, double staining clearly showed that numerous VIP+/AChE-axons were distributed over these arterial regions where VIP+/AChE+ or AChE+/VIP- axons were sporadic or often lacking. The finding that nerve bundles accompanying the ICA within the carotid canal contained abundant VIP+/AChE- nerve cells suggests that cerebrovascular VIP-IR nerves in the quail have their major source at these neurons and enter the cranial cavity through the CCA. Another significant finding was that a small number of nerve cells, which were mostly stained for AChE alone and occasionally for VIP alone or both, occurred in the major arteries located more rostral than the middle AR. Thus, the quail cerebral arterial tree, at least the rostral segment of the anterior circulation, is multiply innervated by these three distinct categories of the extracranial and intracranial VIP-IR and AChE-P neurons.

Progesterone-dependent and -independent expression of the multidrug resistance type I gene in porcine granulosa cells.

A primary role of plasma membrane P-glycoprotein (P-gp), encoded by multidrug resistance type I (MDR1), is to protect against naturally occurring xenotoxics. Progesterone (P(4)) profoundly influences MDR1 expression in granulosa cells and luteal cells. Here, P(4) regulation of MDR1 expression was investigated in porcine granulosa cells using the P(4)-mediated promoter activity assay and a P4 receptor (PR) antagonist (RU-486). The promoter activity was measured chronologically for 48 h in cells transfected with the PR response element-containing pGL3. LH could stimulate the promoter activity through endogenous P4, with a maximum activity at 5 h. MDR1 mRNA level was highly maintained at 24-36 h. Conversely, exogenous P4 prolonged the promoter activity to further 10 h, and the high level of MDR1 mRNA was maintained even at 48 h. RU-486 completely inhibited the promoter activity, but the level of MDR1 mRNA rapidly increased in the presence of RU-486. The granulosa cells may become susceptible to RU-486 as a xenotoxic to rapidly express MDR1 for protection against it. These results indicate that MDR1 is expressed in porcine granulosa cells through P4-dependent and -independent regulations.

Development of multidrug resistance type I P-glycoprotein function during in vitro maturation of porcine oocyte.

The present study was performed to evaluate the expression and function of P-glycoprotein (P-gp) encoded the multidrug resistance type I (MDR1) gene, protecting from xenotoxics, in porcine oocyte during in vitro maturation. Cumulus-oocyte complexes (COCs) were cultured to obtain the germinal vesicle (GV), first metaphase and second metaphase (MII) oocytes. The P-gp function was assessed by means of the rhodamine 6G (R6G) efflux from oocytes with P-gp inhibitors such as verapamil and PSC-833. The MDR1 transcript was detected in the GV and MII oocytes by RT-PCR analysis using primer sets based on the human gene. P-gp inhibitors significantly blocked the R6G efflux from the MII oocytes, whereas the reagents were ineffective in the GV oocytes. The R6G efflux from oocytes was accelerated at the MII stage more than at the GV stage. Thus, the MDR1-type P-gp function is poor at the GV stage, but the function improves during oocyte maturation.

Progesterone regulation of the expression and function of multidrug resistance type I in porcine granulosa cells.

P-glycoprotein (P-gp) coded with the multidrug resistance type I (MDR1) is expressed in various normal tissues including ovaries and may function as detoxification and steroid transport. The present study was performed to analyze the expression and function of MDR1 in granulosa cells stimulated with FSH, LH, estradiol-17beta (E) and progesterone (P). The granulosa cells isolated from porcine ovarian follicles were cultured for 24h in a serum-supplemented medium, and then cultured for 48h with the hormones in a serum-free culture medium. MDR1 was highly expressed in large follicles and induced in cultured granulosa cells stimulated with LH as revealed by RT-PCR. Highly expressed MDR1 resulted in the increased P-gp activity. However, FSH had no effect. P significantly increased the MDR1 expression and P-gp activity in the cells stimulated with LH, whereas E had no stimulatory effect. Aminoglutethimide suppressed the MDR1 expression and P-gp activity, but which were completely restored by P. These results indicate that P participates in MDR1 expression and P-gp function of granulosa cells.

Temporal and spatial expression of TGF-beta2 in chicken somites during early embryonic development.

A multifunctional growth and differentiation factor TGF-beta is expressed at various developmental stages, and its principle role may be involvement in organogenesis. The present study was performed to evaluate the temporal and spatial expression of TGF-beta2 mRNA in developing somites of chicken embryos during their early developmental periods. TGF-betas were expressed in various tissues of the whole embryo obtained at stage 26 (5 days of incubation) as revealed by whole-mount in situ hybridization. TGF-beta2 mRNA was predominantly expressed in somites as well as the head, branchial arch, wing buds, and leg buds. TGF-beta2 mRNA first appeared in the rostral somites on E4, and its expression sites expanded to the middle range of somites at stage 26. At stages 29-31 (6-7 days), expression in the rostral somites disappeared, and it appeared in the caudal somites. TGF-beta2 expression was also analyzed in sections of the embryo by in situ hybridization. The expression sites of TGF-beta2 were clearly observed in the myotomal somite tips as well as the neural tube. RT-PCR analysis showed that TGF-beta2 expression was very low in the blastocyte stage embryo and thereafter increased linearly in the whole trunk until stage 26. These data indicate that TGF-beta2 may be a regulatory factor participating in the somitogenesis of chicken embryos.

Evaluation of RNA interference in developing porcine granulosa cells using fluorescence reporter genes.

Gene silencing using small interfering RNA (siRNA) may be useful for functional analyses of unidentified genes expressed during cell differentiation. The present study was performed to evaluate RNA interference (RNAi) in porcine granulosa cells stimulated with bovine FSH, by using two fluorescence reporter genes: a plasmid encoding green fluorescent protein (GFP) and a plasmid encoding red fluorescent protein (RFP). The siRNA targeting GFP mRNA sequence (GFP-siRNA) with both plasmids was introduced into cultured cells by lipofection. GFP- and RFP-expressing cells were observed under fluorescence microscopy and analyzed by flow cytometry. Strong fluorescence was observed after introduction of both plasmids into cells. The intensity of green fluorescence generated by GFP was greatly suppressed by introduction of GFP-siRNA, showing an approximate 70% decrease in the ratio of green to red fluorescence. Consequently, we concluded that gene silencing by siRNA can be used to analyze the functions of genes of interest during differentiation of porcine granulosa cells.