Localization of putative pituitary stem/progenitor cells in female dairy cattle.

Research on sex-determining region Y-box 2 (SOX2)-positive pituitary stem/progenitor cells, as a source of hormone-producing cells, is progressing rapidly in rodents. However, the stem/progenitor cells supplying hormone-producing cells that are essential for growth, reproduction, and lactation in bovines have not yet been identified. In this study, we characterized SOX2-positive cells in the pituitary gland of dairy cattle (Holstein heifers) after sexual maturity. Immunofluorescence analysis revealed that the localization pattern of SOX2-positive cells in the dairy cattle pituitary gland was similar to that observed in the rodent pituitary gland; the marginal cell layer (MCL), dense cell clusters, and single cells scattered in the parenchyma of the anterior lobe. Furthermore, most of the SOX2-positive cells were positive for the pituitary stem/progenitor cell niche markers E-cadherin and cytokeratin 8+18, which have been reported in rodents. In addition, in the MCL of the anterior lobe, there was a subpopulation of SOX2-positive cells positive for paired-related homeobox 1 and 2, whereas negative for S100ß. Moreover, in the parenchyma of the anterior lobe, co-localization of SOX2 and pituitary hormones was infrequent. In summary, this study reveals the localization of putative pituitary stem/progenitor cells positive for SOX2 in dairy cattle. These results provide valuable information to support further investigation of cell supply in the dairy cattle pituitary gland.

Nicotine inhibits expression of Prrx1 in pituitary stem/progenitor cells through epigenetic regulation, leading to a delayed supply of growth-hormone-producing cells.

OBJECTIVE: Nicotine, a toxic component of smoking, adversely affects animal growth and reproduction by decreasing secretion of anterior pituitary hormones. However, it has not been clarified whether nicotine inhibits the supply of endocrine cells in the pituitary gland. The present study investigated short- and long-term effects of persistent nicotine exposure on the pituitary glands of young animals. DESIGN: Three-week-old male Wistar rats were exposed to nicotine (1 mg/kg body weight/day) for 7 days, and gene expression, cell numbers, and DNA methylation status were analyzed on the following day and 4 weeks after final treatments. RESULTS: The expression level of the stem cell marker Sox2 was not changed by nicotine exposure throughout the experiment. On the other hand, nicotine inhibited expression of a progenitor cell marker, Prrx1, and growth hormone (Gh). Immunohistochemical analysis showed that the SOX2-positive cells positive for PRRX1 in nicotine-treated groups decreased to 61% (4-week-old) and 70% (8-week-old) of the saline-treated controls. In addition, the proportion of GH-positive cells in nicotine-treated group was 14% lower than that of saline-treated controls. Furthermore, first intron hypermethylation of Prrx1 was detected by a bisulfite sequence of genomic DNA from the anterior lobe of the rat pituitary gland. CONCLUSIONS: We show that persistent nicotine exposure in young animals inhibits expression of Prrx1 in pituitary stem/progenitor cells through epigenetic regulation, leading to a delayed supply of GH-producing cells.

Reversal effects of low-dose imatinib compared with sunitinib on monocrotaline-induced pulmonary and right ventricular remodeling in rats.

High-dose imatinib reverses cardiopulmonary remodeling but adverse effects limit its clinical use. Efficacy of the multi-kinase inhibitor sunitinib remains questionable. We compared anti-remodeling effects of imatinib with sunitinib on monocrotaline-induced right ventricular (RV) hypertrophy and pulmonary arterial remodeling in rats, focusing on a lower dose. Fourteen days after monocrotaline injection, oral gavage of imatinib (5, 15, or 50mg/kg), sunitinib (0.3, 1, 3, or 10mg/kg), or water for 14days was started. RV hypertrophy and b-type natriuretic peptide mRNA levels were significantly and dose-dependently reduced, much greater in imatinib- than sunitinib-treated groups. Imatinib normalized muscularization of 20-50µm intra-acinar pulmonary arteries more significantly than sunitinib. At transcript levels, sunitinib significantly upregulated pulmonary nestin, and downregulated platelet-derived growth factor receptor beta (PDGFR-ß), fibroblast growth factor receptor 1, vascular endothelial growth factor receptor-2 and vascular endothelial growth factor (VEGF)-A, but not Raf-1 proto-oncogene serine/threonine kinase mRNAs. Sunitinib also suppressed VEGF-A, but not phosphorylated extra-cellular-signal-related kinase (ERK)-1/2 protein expression. The sole PDGFR-ß antagonism of imatinib resulted in significant Raf-1 mRNA and phosphorylated ERK-1/2 protein downregulation, suggesting that the equivocal reversal effect of sunitinib may be due to its VEGF signaling inhibition in the lung. Imatinib's greater dose-dependent reversal on cardiopulmonary remodeling may make a low dose suitable for PAH treatment.

BNIP3 expression in bovine follicle and corpus luteum.

BNIP3 (BCL2/adenovirus E1B nineteen kilodalton interacting protein-3), a member of the BCL2 family, is activated under hypoxic conditions and induces apoptosis or mitochondrial autophagy for adapting cells to hypoxia. The physiological roles of BNIP3 in the mammalian ovary are still unclear. In order to understand the role of BNIP3 in the bovine ovary, we examined its mRNA and protein expressions of BNIP3 in follicular granulosa cells and corpus luteum (CL). BNIP3 mRNA and protein expressions in granulosa cells from large follicles (>10 mm) at the follicular stage were much higher than those in small follicles (2-8 mm). BNIP3 mRNA and protein expressions in the CL peaked at the early luteal stage. In bovine granulosa cells cultured for 6 hr under hypoxia (3% O2) and normoxia (20% O2), BNIP3 mRNA expression was higher under hypoxia. These results of the present study suggest that BNIP3 has some roles in luteal formation in the bovine ovary, and that the highly expressed BNIP3 in the granulosa cells from large follicles at the follicular stage is related to the roles of BNIP3 in the luteal formation.

Spatial distribution of osteoblast activating peptide in the rat stomach.

Osteoblast activating peptide (OBAP) was previously reported to be expressed in the rat stomach and to have a vital role in osteogenesis, but its distribution in rat stomach has not been determined. Thus, the aim of the present study was to identify the cell types expressing OBAP in the rat stomach. The stomachs of twelve 10-to-11-week-old male Jc1:SD rats were used. Samples were collected for immunohistochemistry, immunoelectron microscopy and dot blot assay. Immunohistochemical investigation revealed that OBAP was distributed mainly in parietal cells without any expression in chief cells, X/A-like cells or enterochromaffin-like cells. Moreover, OBAP-immunopositive cells were observed mainly in the upper and lower parts of the gastric gland. Significantly high optical density of immunopositive cells was observed in the upper and lower gastric gland regions. The dot blot assay confirmed that OBAP is secreted by parietal cells and that it is present in the gastric gland lumen. Immunoelectron microscopy demonstrated that OBAP was confined to the mitochondrial inner membrane within parietal cells and that the number of mitochondria in the upper and lower parts of the gastric epithelium was significantly larger than the number in the middle part of the gastric epithelium. Based on the results, it was concluded that OBAP is mainly produced by mitochondria of parietal cells in the upper and lower parts of the gastric epithelium. Moreover, the presence of OBAP in the gastric gland lumen suggests an exocrine mechanism of release.

Expression of anti-Müllerian hormone and its type II receptor in germ cells of maturing rat testis.

This work aimed to clarify the expression and roles of anti-Müllerian hormone (AMH) and its type 2 receptor (AMHR2) in seminiferous tubules of maturing rat testes. By quantitative RT-PCR, we determined the relative expressions of Amh, Amhr2, Scp1, Rsbn1, Ngfr, and Rhox5 in rat testes aged 5-49 days (d), and in germ cells and Sertoli cells isolated from 21d testes. Smad 1,5 and 8 expressions were also determined in 21d testes and isolated germ cells. Moreover, we performed in situ hybridization (ISH) of Amh and Amhr2 in 21d testes, and immunohistochemical staining (IHCS) in 10, 15 and 21d testes using antibodies of AMH and AMHR2. In 21d testes, expression of the spermatocyte specific gene, Scp1, increased but that of the round spermatid specific gene, Rsbn1, was faint. By ISH and IHCS, expressions of AMH and AMHR2 were strongly observed in spermatocytes of 21d testes, but not in spermatogonia. In 21d testes, expressions of immature Sertoli cell specific gene, Ngfr, and mature Sertoli cell specific gene, Rhox5, were observed. IHCS confirmed the presence of AMH and AMHR2 in Sertoli cells. Smad 1, 5 and 8 were highly expressed in 21d testes and isolated germ cells. These results indicate that not only immature Sertoli cells but also spermatocytes express AMH and AMHR2 in maturing testes. In this study, we first clarified that spermatocytes coexpressed AMH and AMHR2 in rats. We speculated that AMH produced by spermatocytes and Sertoli cells binds AMHR2 of spermatocytes and acts through SMADs.

Expression and intracellular localization of TBC1D9, a Rab GTPase-accelerating protein, in mouse testes.

Membrane trafficking in male germ cells contributes to their development via cell morphological changes and acrosome formation. TBC family proteins work as Rab GTPase accelerating proteins (GAPs), which negatively regulate Rab proteins, to mediate membrane trafficking. In this study, we analyzed the expression of a Rab GAP, TBC1D9, in mouse organs and the intracellular localization of the gene products. Tbc1d9 showed abundant expression in adult mice testis. We found that the Tbc1d9 mRNA was expressed in primary and secondary spermatocytes, and that the TBC1D9 protein was expressed in spermatocytes and round spermatids. In 293T cells, TBC1D9-GFP proteins were localized in the endosome and Golgi apparatus. Compartments that were positive for the constitutive active mutants of Rab7 and Rab9 were also positive for TBC1D9 isoform 1. In addition, TBC1D9 proteins were associated with Rab7 and Rab9, respectively. These results indicate that TBC1D9 is expressed mainly in spermatocytes, and suggest that TBC1D9 regulates membrane trafficking pathways related to Rab9- or Rab7-positive vesicles.

Tumor suppressor candidate TUSC3 expression during rat testis maturation.

Analysis of microarray data obtained by comparing gene expression between 2-week-old infant and 7-week-old mature SD rat testes revealed novel targets involved in tumor suppression. Reverse-transcription polymerase chain reaction and Northern blotting indicated that Tusc3 gene expression was upregulated in the normal maturing testis and prostate and other organs such as the cerebrum and ovary. Tumor suppressor candidate 3 protein expression was detected in these same organs at a size of about 40 kDa, in accord with the predicted molecular size. In situ hybridization and immunohistochemistry showed that mRNA and protein localization were prevalent in the testis spermatocytes and interstitial cells such as the Leydig cells, as well as prostate epithelial cells. These data suggest that TUSC3 is deeply involved in spermatogenesis in the testis, inducing sperm differentiation and maturation, and plays a role in normal prostate development and tumor suppression.

Antiviral and antiproliferative effects of canine interferon-λ1.

Interferon (IFN)-λs, members of the type III IFN group, were recently identified in several vertebrates. Although IFN-λs have the potential to be utilized as antiviral and antitumor agents in veterinary medicine, the biological properties of IFN-λs have not yet been studied in companion animals. In this study, we analyzed the expression of canine IFN-λs and their receptors, produced a recombinant canine IFN-λ1 protein, and investigated its antiviral and antiproliferative activities using a canine kidney epithelial cell line, MDCK cells. MDCK cells were found to express type III IFN molecules, IFN-λ1 and IFN-λ3, and the receptors, IFNλR1 and IL10R2. IFN-λ1 was induced faster than IFN-λ3 by stimulation with poly (I:C). His-tagged IFN-λ1 protein expressed in Escherichia coli inhibited cytolytic plaque formation by influenza A virus infection, and induced the expression of interferon-stimulated genes, Mx1 and OAS1, in MDCK cells. In addition, recombinant IFN-λ1 inhibited the proliferation of MDCK cells slightly. These effects were observed in a dose-dependent manner. These results indicate that canine IFN-λ1 has antiviral effect, and suggest the potential applicability of canine IFN-λ1 as a therapeutic agent.

Rat stem-cell leukemia gene expression increased during testis maturation.

We found that stem-cell leukemia (SCL), also known as T cell acute-lymphocytic leukemia (Tal-1) gene expression, was upregulated in the maturing rat testis. Strong expression of Tal-1 was detected in the normal maturing rat testis by Northern blotting. Western blotting revealed the protein size to be about 34 kDa. Protein expression was wide-spread in spermatocytes, spermtids and spermatogonia in accordance with the seminiferous epithelium cycle, as determined by an analysis of immunohistochemistry. Gene expression of Tal-1 regulatory gene, NKX3.1, was negatively correlated with Tal-1 expression. Human Tal-1 expression in the maturing testis as well as in bone marrow was observed, which suggests that the gene product is a novel cancer-testis antigen candidate. Taken together, TAL-1 may be involved in cell division, morphological changes, and the development of spermatogenic cells in the normal rat testis.

Expression of a novel sphingosine 1-phosphate receptor motif protein gene in maturing rat testes.

We screened a novel sphingosine 1-phosphate receptor type 5 motif-containing gene, LOC290876, from maturing rat testes by differential display. Gene expression was testis-specific, increased at week 7, and continued for 15 weeks. PCR analysis clarified two gene transcript isoforms, which were expressed at the same level in all samples detected in Northern blot. The deduced amino acid sequences of the two isoforms revealed differences in carboxyl terminal sequences. Gene and protein expression in the testes was dominant in the spermatocytes, and protein expression was localized to the nucleus. Taken together, these findings suggest that the LOC290876-encoded gene product is not involved in sphingosine signaling, but has distinct roles in the nucleus during the processes of spermatocyte maturation and meiosis producing spermatids.

Regulation of rat tetratricopeptide repeat domain 29 gene expression by follicle-stimulating hormone.

We screened the gene that encodes tetratricopeptide repeat domain 29 (Ttc29) in the maturing rat testis. Gene expression was determined by Northern blotting of 7-week-old rat testes, and a strong signal was detected close to the 18S rRNA band in addition to two weak high-molecular-weight signals. In situ hybridization revealed that Ttc29 was expressed primarily in the spermatocytes. We evaluated the effect of gonadotropin on Ttc29 expression using hypophysectomized rats. The pituitary was removed from 3-week-old rats, gonadotropin was injected at 5 weeks, and Ttc29 expression was determined at 7 weeks. Although testicular development and hyperplasia of interstitial cells were observed following chorionic gonadotropin treatment after hypophysectomy, Ttc29 expression was upregulated by treatment with follicle-stimulating hormone. Ttc29 encodes axonemal dynein, a component of sperm flagella. Taken together, these data indicate that axonemal dynein expression starts in the spermatocytes and is regulated by follicle-stimulating hormone.

Biological effects of chicken type III interferon on expression of interferon-stimulated genes in chickens: comparison with type I and type II interferons.

Interferons (IFNs) are key mediators that activate host defense mechanisms against viruses. The recently identified mammalian Type III IFN has biological effects similar to type I IFN. However, the biological effects of type III IFN have not yet been characterized in birds. We compared the effects of chicken type III IFN (IFN-λ) with type I (IFN-ß) and type II (IFN-γ) IFNs on IFN-stimulated genes (ISGs) using recombinant proteins expressed in Escherichia coli. Recombinant chicken IFN-λ inhibited influenza virus replication and induced the mRNA expression of the ISGs, Mx and OAS, in chicken embryonic fibroblasts (CEFs) in a dose-dependent manner. However, the effective dose of IFN-λ was higher than that of IFN-ß and IFN-γ. Furthermore, the effect of IFN-λ on induction of Mx and OAS was lesser than that of IFN-ß, but comparable to that of IFN-γ. These results indicate that chicken IFN-λ has the potential to induce ISGs and inhibit viral replication in chicken cells.

Expression of the centrosomal colon cancer autoantigen gene during spermatogenesis in the maturing rat testis.

We analyzed the gene and protein expression of serologically defined colon cancer antigen 8. Gene expression was upregulated in the maturing rat testis, and was localized to the spermatocytes. Protein was detected in the spermatids and at the sites of mRNA expression. Specific expression of colon cancer antigen 8 was observed in the maturing rat testis.

Expression of rat sperm flagellum-movement associated protein genes under 2,3,7,8-tetrachlorodibenzo-p-dioxin treatment.

We analyzed the gene expression of sperm flagellum-movement associated proteins, namely, adenylate kinase domain containing protein (RGD1303144) and outer dense fiber protein 1. These gene expressions were up-regulated in a maturing rat testis, and RGD1303144 gene was expressed dominantly in spermatocytes. Although 2,3,7,8-tetrachlorodibenzo-p-dioxin administration reduced sperm motility, these gene expressions were not affected.

Automated SNPs typing system based on the Invader assay.

Typing of single nucleotide polymorphisms (SNPs) has advantages in forensic DNA identification because SNPs are abundant in genomic DNA and are easily detected using an automated high-throughput system. In this study, we examined the effectiveness of an automated multiplex SNPs typing system based on the "Invader assay". Using this system, multiplex SNPs typing is completed in 40 min without any complex procedures. The typing results were confirmed by direct sequencing, and no inconsistencies were detected between the sequencing results and the typing results. The population data of 21 SNPs from 113 Japanese indicates that the matching probability is about 1.3 x 10(-9). In our experiment, all of 21 SNPs were correctly detected from degraded samples from which typing of short tandem repeat markers was difficult.

Expression of the Ha-ras suppressor family member 5 gene in the maturing rat testis.

We analyzed the gene expression of Ha-ras suppressor family member 5 (Hrasls5), which is considered to modulate the Ha-ras signaling cascade, from maturing rat testis. Expression was detected primarily in the spermatocytes in the maturing rat testis. The Hrasls5 gene product might function as a tumor suppressor as well as in spermatogenesis, as deduced from its amino acid sequence.

Cloning and expression of a gene encoding gallerimycin, a cysteine-rich antifungal peptide, from eri-silkworm, Samia cynthia ricini.

A cDNA clone encoding gallerimycin was isolated from larval fat body of immunized Samia cynthia ricini and named as Scr-gallerimycin. In naive larvae, no gene expression was detected, but strongly induced in fat body and hemocytes following immune challenge with bacteria or entomopathogenic fungus Beauveria bassiana. Strong expression of the gene was also induced by injection of peptidoglycan and zymosan, but very weakly by non-pathogenic fungus Aspergillus oryzae. Analysis of the sequence upstream from the cDNA shows the presence of motifs homologous to binding sites for NF-kappaB, C/EBP and CRE-BP1.

Induction of tyrosine hydroxylase gene expression by bacteria in the fat body of eri-silkworm, Samia cynthia ricini.

A cDNA clone encoding tyrosine hydroxylase (TH) was isolated from larval fat body of immunized Samia cynthia ricini. In naive larvae, the TH gene was expressed only in the brain, but strongly induced in fat body and hemocytes after injecting UV-killed bacteria. The induction of the gene was rather short-lived compared to that of antibacterial protein genes, reaching the maximum levels 6h after bacterial challenge, and then quickly diminished. A strong induction of the gene expression was caused by both Gram-negative and positive bacteria and zymosan, but little if any by soluble peptidoglycan or lipopolysaccharide. A possible role of TH in the fat body of bacteria-challenged larvae would be to supply catecholamines as the substrate for phenoloxidase leading to melanization, working together with dopa decarboxylase.

Three peptidoglycan recognition protein (PGRP) genes encoding potential amidase from eri-silkworm, Samia cynthia ricini.

Three cDNA clones encoding peptidoglycan recognition proteins (PGRP-B, -C and -D) were isolated from larval fat body of immunized Samia cynthia ricini. The deduced amino acid sequences show high homology to each other and also to Drosophila PGRP-LB, but rather lower homology to all of the known lepidopteran PGRPs including Samia PGRP-A, a receptor-type PGRP. The three PGRPs conserve the five amino acid residues which form the catalytic site of N-acetylmuramoyl L-alanine amidase as in Drosophila LB. The PGRP-C and -D genes were silent in naive larvae, but strongly induced in fat body by an injection of peptidoglycan. PGRP-B gene, in contrast, constitutively expressed at high levels in naive midgut, and the gene was weakly induced in fat body after injection of peptidoglycan.