Ontogeny of erythropoietin receptor mRNA expression in various tissues of the foetal and the neonatal pig

Erythropoietin receptor (EPOR) mRNA expression in liver, spleen, bone marrow and testes of foetal and neonatal pigs was analysed using a real-time RT-PCR assay. The results showed that early in the foetal life, EPOR expression is greatest in the liver. Later in foetal life, the spleen has the greatest expression of EPOR, whereas at 2 weeks after birth, the main expression of EPOR is found in the bone marrow. These findings contradict our earlier hypothesis that erythropoietin (EPO) acting in a paracrine fashion can account for an extensive erythropoiesis at birth, a point of time when plasma EPO concentrations are low. Results presented in the present paper suggest that the spleen or, alternatively, the bone marrow is able to respond to very low concentrations of circulating EPO around the time of birth. The testes were found to express significant amounts of EPOR. Since EPO mRNA has previously been found in the testes, a paracrine function of EPO may exist in this organ.     

猪颈前神经节脊髓颈部和胃内Leptin长形受体mRNA的分布定位

用原位杂交法研究了10头长白猪(n=5)和梅山猪(n=5)颈前神经节、脊髓颈部和胃内Ob—Rb mRNA的分布定位。实验结果表明，Ob—Rb mRNA标记神经元位于长白猪和梅山猪颈前神经节、脊髓颈部和胃内。在颈前神经节，Ob—Rb mRNA标记神经元散在分布，胞体呈圆形或卵圆形。在脊髓颈部，Ob—Rb mRNA标记神经元分布于背侧角和腹侧角，以背侧角分布密集。在胃内，Ob—Rb mRNA标记细胞分布于黏膜层和黏膜下层。长白猪和梅山猪上述结构内Ob—Rb mRNA的分布定位无明显差异。     

The expression of histamine H4 receptor mRNA in the skin and other tissues of normal dogs

The histamine 4 (H₄) receptor was first cloned and characterized in 2000 using the human H₃ receptor DNA sequence. The H₄ receptor has been shown to participate in various aspects of inflammation, such as chemotaxis, upregulation of adhesion molecule expression and modulation of cytokine secretion. The primary goal of this study was to determine whether H₄ receptor mRNA is expressed in normal canine skin by performing an RT‐PCR. An additional goal was to determine the expression of this receptor in the colon, liver, spleen and kidney. Tissues were collected from five healthy, young‐adult pit bull dogs. Samples were immediately placed in RNAlater^® solution and stored at ?20°C until processed. The amplified products in all skin samples in addition to the colon, liver, spleen and kidney (variable expression) had the expected size of 400–500 bp. The sequenced amplicons matched the National Center for Biotechnology Information published sequence for the canine H₄ receptor. The study results showed that canine normal skin expresses the H₄ receptor mRNA. Further studies using immunohistochemistry should be conducted to demonstrate the expression of the H₄ receptor at the protein level and to localize the expression of this receptor in the skin.     

猪ADSL基因克隆及其在部分组织中的mRNA定量表达

试验以通城猪为研究对象,在对猪ADSL基因全长编码区cDNA片段进行克隆、序列测定、与GenBank中已收录的其他14种动物的ADSL全长编码区序列进行同源性分析,并构建系统发生树的基础上,采用实时(相对)定量PCR方法对刚出生和65日龄两个不同生长发育时期的通城猪心、肝、肾脏、背最长肌4种组织的ADSL基因表达谱进行分析。结果表明,所克隆的ADSL基因片段大小为1 548 bp(GenBank登录号:GU249574),其中包含一长为1 455 bp的完整阅读框,通过核酸序列分析发现,该基因编码484个氨基酸;与GenBank中已收录的猪ADSL基因序列同源性最高,达99.9%;与恒河猴和黑猩猩的序列同源性最低,分别为66.3%和41.3%;15种动物的20条ADSL全长CDS序列聚类分布为两个主支。刚出生时的通城猪ADSL基因在心、肝、肾脏组织的mRNA表达水平均高于65日龄通城猪的心、肝、肾脏组织中的mRNA表达;然而,其背最长肌的ADSL基因mRNA表达水平则低于65日龄的通城猪。本结果为猪ADSL基因的生物学功能研究和遗传进化研究提供了分子依据。     

Molecular cloning and tissue distribution of a short form chicken leptin receptor mRNA

In mammals, alternative splicing of the leptin receptor (LEPR) produces several C-terminal truncated isoforms that are believed to play a role in the transport, cellular internalisation and degradation of the hormone leptin. The chicken leptin receptor (chLEPR) is similar to its mammalian counterparts in terms of its intron/exon structure and conserved motifs. However, it is unknown whether the chLEPR also undergoes alternative splicing. To test this, structural analysis of intron 19 of the chLEPR, equivalent to the intron in which alternative splicing occurs in mammals, was combined with 3'-rapid amplification of cDNA ends (3'-RACE) to search for chLEPR splice variants. A 44-amino acid alternative exon 20 was identified that is spliced to generate a short isoform of the chLEPR (chLEPR-SF). Comparative sequence analysis of intron 19 identified two regions that are highly conserved between the chicken and mammals, indicating their possible importance as intronic elements in the regulation of alternative splicing of the LEPR in vertebrates. Tissue expression of the chLEPR-SF was lower and more restricted than that of the chLEPR long isoform. Collectively these data demonstrate that the chLEPR is alternatively spliced to produce at least one short isoform, as is the case in mammals.     

Preliminary report on the expression of leptin and leptin receptor (ObR) in normal, hyperplastic and neoplastic canine mammary tissues

Leptin and its receptor (ObR) expression were investigated by immunohistochemistry in normal, hyperplastic and neoplastic canine mammary tissues and related to clinical-pathological features. Leptin expression was detected in healthy mammary tissues, adenosis and in benign mammary tumours and was lower in ductal hyperplasias and malignant tumours. A high percentage of ObR-positive cells were present in adenosis, benign tumours and in complex carcinomas, while ObR expression was lower in healthy mammary tissues, in ductal hyperplasias and in a large part of invasive mammary carcinomas. Our data demonstrated that cancer cells expressed at low level leptin and ObR in canine mammary tumours with a more aggressive behaviour, as well as in lymph node metastases. Consequently, leptin and ObR expressions in this species resulted to be not associated with a reduced overall survival.     

Sexual maturation in hens is not associated with increases in serum leptin and the expression of leptin receptor mRNA in hjpothalamus

Background： In mammals, leptin is an attractive candidate for mediating the metabolic signal and the reproductive function via the specific receptor in hypothalamus. However, till now, the role of leptin on reproduction in birds is less well established. This experiment was conducted to elucidate the role of leptin on the onset of reproduction in bird, as a first step, to detect the changes of peripheral leptin and leptin receptor mRNA expression in hypothalamus between mature and immature hens at the same age. 120 ISA brown pullets at D60 were allocated randomly into two groups, long light （LL） group being raised under artificial light regimes with incrementally increased light phase （from 8 L：]6D to 14 L：]2D） and short light （SL） group raised on consistent light （8 L：16D） for 12 wk. Results： The results showed that pullets in LL group reached sexual maturation 15 d earlier than those in SL group. Serum E2 showed a significant increase with age, but no difference was observed between two groups. Serum leptin concentration decreased significantly from D112 to D136 in LL, and was markedly higher in LL group than that in SL at D112, while there was no significant difference between two groups at D136. Leptin receptor and GnRH-I mRNA expression in hypothalamus were significantly increased with age, yet there was no significant difference between SL and LL chickens at the same age. The expression of FSH-13 and LH-13 mRNA in pituitary was increased with age but did not show significant difference between LL and gland, and decreased from D112 to D136 in LL but not groups at the same age. SL group. GnfiH-I mRNA expression was very rich in pinea n SL group, and there was no difference between two Conclusions： These results indicate that the earlier sexual maturation in hens induced by long-light regime is not accompanied with an increase in serum leptin or leptin receptor gene expression in hypothalamus, or genes expression in HPG axis.     

Sexual maturation in hens is not associated with increases in serum leptin and the expression of leptin receptor mRNA in hypothalamus

Background

In mammals, leptin is an attractive candidate for mediating the metabolic signal and the reproductive function via the specific receptor in hypothalamus. However, till now, the role of leptin on reproduction in birds is less well established. This experiment was conducted to elucidate the role of leptin on the onset of reproduction in bird, as a first step, to detect the changes of peripheral leptin and leptin receptor mRNA expression in hypothalamus between mature and immature hens at the same age. 120 ISA brown pullets at D60 were allocated randomly into two groups, long light (LL) group being raised under artificial light regimes with incrementally increased light phase (from 8 L:16D to 14 L:12D) and short light (SL) group raised on consistent light (8 L:16D) for 12 wk.

Results

The results showed that pullets in LL group reached sexual maturation 15 d earlier than those in SL group. Serum E₂ showed a significant increase with age, but no difference was observed between two groups. Serum leptin concentration decreased significantly from D112 to D136 in LL, and was markedly higher in LL group than that in SL at D112, while there was no significant difference between two groups at D136. Leptin receptor and GnRH-I mRNA expression in hypothalamus were significantly increased with age, yet there was no significant difference between SL and LL chickens at the same age. The expression of FSH-β and LH-β mRNA in pituitary was increased with age but did not show significant difference between LL and SL group. GnRH-I mRNA expression was very rich in pineal gland, and decreased from D112 to D136 in LL but not in SL group, and there was no difference between two groups at the same age.

Conclusions

These results indicate that the earlier sexual maturation in hens induced by long-light regime is not accompanied with an increase in serum leptin or leptin receptor gene expression in hypothalamus, or genes expression in HPG axis.     

Comparing mRNA levels of genes encoding leptin,leptin receptor,and lipoprotein lipase between dairy and beef cattle

Body weight and fat mass vary distinctly between German Holstein (dairy cattle) and Charolais (beef cattle). The aim of this study was to determine whether the expression of the obese (Ob) gene and lipoprotein lipase (LPL) gene in fat tissues and expression of the long isoform leptin receptor (Ob-Rb) gene in the hypothalamus were different between these two cattle breeds. Body weight and the area of longissimus muscle cross-section of German Holstein were lower (P<0.001), while body fat content, as well as the omental and perirenal fat mass were higher (P<0.001), compared to Charolais. Plasma insulin and leptin levels between two cattle breeds were determined by radioimmunoassay. Compared to Charolais, plasma insulin concentrations were significantly higher (P<0.01), and plasma leptin levels were tended to be higher (P<0.1) in German Holstein. Ob mRNA levels in subcutaneous and perirenal fat depots, but not in the omental fat depot, were significantly higher (P<0.05) in German Holstein than in Charolais. LPL mRNA expression in the perirenal fat depot of German Holstein was greater in abundance than that of Charolais. No significantly different LPL mRNA levels were found in subcutaneous and omental fat depots, and Ob-Rb mRNA levels in the hypothalamus between these two cattle breeds (P<0.05). Both Ob and LPL expression was greater in perirenal and omental fat depots than in the subcutaneous fat depot (P<0.05). Data indicated that in bovine the Ob and LPL gene expression levels in perirenal fats are an important index that is associated with body fat content, while Ob-Rb in hypothalamus is not.     

Predominant expression of growth hormone secretagogue receptor in the caudal lobe of chicken pituitary and parallel developmental increase in expression of pituitary GH and proventriculus ghrelin mRNA

To examine the involvement of ghrelin in growth hormone (GH) synthesis in the chicken pituitary, the regional distribution of GH secretagogue receptor (GHS‐R)/ghrelin receptor was investigated. Quantitative real‐time polymerase chain reaction (Q‐PCR) analysis revealed that the expression levels of GHS‐R and GH mRNA in the caudal lobe were about fourfold and sevenfold higher in the cephalic lobe of 7 day‐old chickens, respectively. Immunohistochemical analysis showed that GHS‐R immunoreactivity was more abundant in the caudal lobe than in the cephalic lobe, as was the case for GH immunoreactivity. By Q‐PCR, parallel increases were observed in the expression levels of ghrelin mRNA in the proventriculus and GH mRNA in the pituitary from embryonic day 17 to day 7 after hatching, whereas no significant change was found in the expression levels of GHS‐R mRNA in the pituitary during this period. These results suggest that proventriculus‐derived ghrelin may participate in pituitary GH synthesis by acting on its receptor during late embryonic development and the early post‐hatching period in chickens.     

Quantitative analysis of beta-adrenergic receptor subtypes in pig tissues

The density and distribution of beta1- and beta2-adrenergic receptors (betaAR) in porcine adipocytes, skeletal muscle, heart, lung, and liver were investigated using competitive displacement of ligand binding with subtype-selective ligands. Three experimental approaches were used to estimate the distribution of betaAR subtypes in adipocytes. Two approaches involved simultaneous linear regression analysis of multiple competitive displacement curves with the beta1AR-selective antagonist CGP 20712A and the beta2AR-selective ligand BRL 37344. For the third approach, radioligand saturation assays were perfomed using a concentration of CGP 20712A that completely blocked the beta1AR. All three approaches indicated the presence of multiple betaAR subtypes in porcine adipocytes and gave similar estimates for the proportion of these subtypes. Saturation assays in the presence of the beta1AR blocker CGP 20712A were conducted to determine the distribution of the betaAR subtypes in skeletal muscle, heart, lung, and liver. The proportions of the beta1AR and beta2AR were 81:19, 59:41, 72:28, 58:42, and 50:50 for adipose, skeletal muscle, heart, lung, and liver, respectively. These estimates based on receptor protein were consistent with published estimates of mRNA abundance in pig tissues but differ from estimates for other species. The predominance of beta1AR in adipocytes and skeletal muscle may contribute to the reduced efficacy of select betaAR agonists in pigs compared to other species because most of the ligands evaluated in growth studies are purported to be beta2AR selective. The density of the betaAR varied among tissues in the following order: heart = lung > adipocytes > skeletal muscle or = liver.     

Biology of leptin in the pig

The recently discovered protein, leptin, which is secreted by fat cells in response to changes in body weight or energy, has been implicated in regulation of feed intake, energy expenditure and the neuroendocrine axis in rodents and humans. Leptin was first identified as the gene product found deficient in the obese ob/ob mouse. Administration of leptin to ob/ob mice led to improved reproduction as well as reduced feed intake and weight loss. The porcine leptin receptor has been cloned and is a member of the class 1 cytokine family of receptors. Leptin has been implicated in the regulation of immune function and the anorexia associated with disease. The leptin receptor is localized in the brain and pituitary of the pig. The leptin response to acute inflammation is uncoupled from anorexia and is differentially regulated among swine genotypes. In vitro studies demonstrated that the leptin gene is expressed by porcine preadipocytes and leptin gene expression is highly dependent on dexamethasone induced preadipocyte differentiation. Hormonally driven preadipocyte recruitment and subsequent fat cell size may regulate leptin gene expression in the pig. Expression of CCAAT-enhancer binding protein (C/EBP) mediates insulin dependent preadipocyte leptin gene expression during lipid accretion. In contrast, insulin independent leptin gene expression may be maintained by C/EBP auto-activation and phosphorylation/dephosphorylation. Adipogenic hormones may increase adipose tissue leptin gene expression in the fetus indirectly by inducing preadipocyte recruitment and subsequent differentiation. Central administration of leptin to pigs suppressed feed intake and stimulated growth hormone (GH) secretion. Serum leptin concentrations increased with age and estradiol-induced leptin mRNA expression in fat was age and weight dependent in prepuberal gilts. This occurred at the time of expected puberty in intact contemporaries and was associated with greater LH secretion. Further work demonstrated that leptin acts directly on pituitary cells to enhance LH and GH secretion, and brain tissue to stimulate gonadotropin releasing hormone secretion. Thus, development of nutritional schemes and (or) gene therapy to manipulate leptin secretion will lead to practical methods of controlling appetite, growth and reproduction in farm animals, thereby increasing efficiency of lean meat production.     

Androgen receptor mRNA expression in the bovine ovary

Previous studies have shown that androgen receptor (AR) is expressed in granulosa cells of healthy, growing ovarian follicles in rats and primates. However, AR expression in the bovine ovary has not been examined. Therefore, a 346-base pair segment of the bovine AR was cloned and sequenced. Using a ribonuclease protection assay, AR expression was detected in total RNA from bovine ovarian cortex. Expression (absence or presence) of AR mRNA was detected by in situ hybridization in bovine ovarian cortex. Follicles (n = 32) were classified as follows: type 1 (1 layer of flattened granulosa cells), type 2 (1-1.5 layers of cuboidal granulosa cells), type 3 (2-3 layers of granulosa cells), type 4 (4-6 layers of cuboidal granulosa cells and formation of thecal layer), and type 5 (>6 layers of cuboidal granulosa cells, defined theca layer, and antrum formation). Frequency of AR mRNA expression increased (P < 0.001) as follicles entered the growing pool. Expression of AR mRNA was absent in type 1 follicles (n = 8), but present in the granulosa cells of 41% of type 2 follicles (n = 12). In types 3-5 follicles, AR mRNA expression was present in granulosa cells of 100% of follicles examined (n = 4, 4, and 4, respectively) and was greater than type 1 follicles (P = 0.002). These data provide evidence of AR mRNA expression in bovine follicles and suggest that AR mRNA increases during early follicle development.     

Effects of an intravenous injection of NPY on leptin and NPY-Y1 receptor mRNA expression in ovine adipose tissue

Neuropeptide Y (NPY) is highly expressed in hypothalami of undernourished and genetically obese animals, and is a potent regulator of food intake and reproduction. Leptin, a protein expressed by adipocytes, has been reported to reduce hypothalamic NPY expression. We recently detected (by ribonuclease protection assay [RPA]) expression of the NPY receptor subtype Y1 (but not Y2) mRNA in adipose tissue. Based on these observations we hypothesized that NPY-Y1 receptors in adipose may represent a peripheral mechanism by which NPY can regulate leptin expression in a direct and rapid manner. To test this hypothesis, adipose samples were biopsied from the tailhead region of 48 ± 3 kg wether lambs immediately before and 30 min after a single intravenous injection of 50 μg porcine NPY (“treated” animals, n = 5), or vehicle (“control” animals, n = 4). Injection of NPY resulted in an increase in expression (P = 0.013; as measured by RPA) of both leptin and NPY-Y1 mRNA. In treated animals, negative correlations were found between response in leptin expression and body weight (r = −0.82, P = 0.092), and between leptin response and initial leptin mRNA levels (r = −0.81, P = 0.097). These data provide evidence of a peripheral mechanism by which NPY may regulate adipocyte expression of both leptin and NPY-Y1 receptor mRNA.