Effect of recombinant growth hormone on expression of growth hormone receptor,insulin-like growth factor mRNA and serum level of leptin in growing pigs

Sixteen Large White × Landrace castrated male pigs were allotted into treatment and control group. The treatment group was injected intramuscularly with recombinant porcine growth hormone (rpGH, 4 mg d⁻¹) and the control group with vehicle for 28 days. Animals were slaughtered 4 h after final injection for liver, longissimus dorsi (LD) muscle and blood sampling. Serum concentration of insulin-like growth factor 1 (IGF-I) and leptin were determined by RIA. The total RNA was extracted from tissues to measure the abundance of growth hormone receptor (GHR), IGF-I mRNA by RT-PCR with 18S rRNA internal standard. Results showed that rpGH enhanced the average daily weight gain by 26.1% (P < 0.05), the serum IGF-I concentration by 70.94% (P < 0.01), decreased serum leptin by 34.8% (P < 0.01). The relative abundance of GHR and IGF-I mRNA in liver were increased by 24.45% (P < 0.05) and 45.30% (P < 0.01), respectively, but no difference of GHR (P > 0.05) and IGF-I mRNA (P > 0.05) in LD between GH treated and control group was found. These results suggest that rpGH can up-regulate hepatic GHR and IGF-I gene expression and improve animal growth. However the effect of rpGH on GHR and IGF-I gene expression are tissue-specific.     

Thyroid hormone receptor subtype specificity for hormone-dependent neurogenesis in Xenopus laevis

Thyroid hormone (T₃) influences cell proliferation, death and differentiation during development of the central nervous system (CNS). Hormone action is mediated by T₃ receptors (TR) of which there are two subtypes, TRα and TRβ. Specific roles for TR subtypes in CNS development are poorly understood. We analyzed involvement of TRα and TRβ in neural cell proliferation during metamorphosis of Xenopus laevis. Cell proliferation in the ventricular/subventricular neurogenic zones of the tadpole brain increased dramatically during metamorphosis. This increase was dependent on T₃ until mid-prometamorphosis, after which cell proliferation decreased and became refractory to T₃. Using double labeling fluorescent histochemistry with confocal microscopy we found TRα expressed throughout the tadpole brain, with strongest expression in proliferating cells. By contrast, TRβ was expressed predominantly outside of neurogenic zones. To corroborate the histochemical results we transfected living tadpole brain with a Xenopus TRβ promoter-EGFP plasmid and found that most EGFP expressing cells were not dividing. Lastly, treatment with the TRα selective agonist CO23 increased brain cell proliferation; whereas, treatment with the TRβ-selective agonists GC1 or GC24 did not. Our findings support the view that T₃ acts to induce cell proliferation in the tadpole brain predominantly, if not exclusively, via TRα.     

Effect of recombinant growth hormone on expression of growth hormone receptor, insulin-like growth factor mRNA and serum level of leptin in growing pigs

Growth hormone (GH) plays an important role in regulation of animal growth, metabolism and lactation[1]. Numerous studies have shown that exogenous somatotropin (ST) can increase average daily weight gain, improve feed efficiency, stimulate protein deposition and muscle growth and decrease lipid accretion rate[1]. The original somatomedin hypothesis suggested that the effect of GH on postnatal growth was mediated by insulin-like growth hormone factor 1 (IGF-I) which was thought to be deriv…     

Molecular cloning and mRNA tissue expression of thyroid hormone receptors in yellow catfish Pelteobagrus fulvidraco and Javelin goby Synechogobius hasta

Thyroid hormones (THs) play a pivotal role in many physiological functions in vertebrates, including fish. Their effects are mediated by thyroid hormone receptors (TRs), which are members of the nuclear hormone receptor superfamily. In this study, full-length cDNA sequences of TRs from yellow catfish Pelteobagrus fulvidraco and Javelin goby Synechogobius hasta were cloned and their mRNA tissue expression profiles were determined. In P. fulvidraco, the validated cDNAs encoding for TRα and TRβ were 1789 and 1848 bp in length, encoding peptides of 401 and 378 amino acid residues, respectively. In addition, a TRβ spliced variant (named P. fulvidraco-TRβv), containing a 60-bp insertion, was detected. In S. hasta, cDNAs encoding for TRαA, TRαB and TRβ were 1827, 2295 and 2258 bp in length, encoding peptides of 401, 409 and 393 amino acid residues, respectively. The phylogenetic analysis revealed that TRα and TRβ cDNAs grouped into two separate clusters with other vertebrate counterparts and two TRα sequences grouped separately, suggesting that the two TRαs derived from paralogous genes that might arise during a teleost-specific genome duplication event. All TR mRNAs were detected in various tissues sampled. The mRNA levels of both TRα and TRβ from P. fulvidraco were the highest in brain, followed by liver, and lowest in heart, intestine, muscle, gill and spleen. However, in S. hasta, TRαA, TRαB and TRβ showed the highest mRNA levels in brain and lowest in muscle. Identification and mRNA tissue expression of TR genes from P. fulvidraco and S. hasta provide an initial step towards understanding their biological roles in the two fish species.     

Organ-specific effects on target binding due to knockout of thyroid hormone receptor α during Xenopus metamorphosis

Thyroid hormone (T3) is essential for normal development and metabolism, especially during postembryonic development, a period around birth in mammals when plasma T3 levels reach their peak. T3 functions through two T3 receptors, TRα and TRβ. However, little is known about the tissue-specific functions of TRs during postembryonic development because of maternal influence and difficulty in manipulation of mammalian models. We have studied Xenopus tropicalis metamorphosis as a model for human postembryonic development. By using TRα knockout (Xtr·thra^tmshi) tadpoles, we have previously shown that TRα is important for T3-dependent intestinal remodeling and hindlimb development but not tail resorption during metamorphosis. Here, we have identified genes bound by TR in premetamorphic wild-type and Xtr·thra^tmshi tails with or without T3 treatment by using chromatin immunoprecipitation–sequencing and compared them with those in the intestine and hindlimb. Compared to other organs, the tail has much fewer genes bound by TR or affected by TRα knockout. Bioinformatic analyses revealed that among the genes bound by TR in wild-type but not Xtr·thra^tmshi organs, fewer gene ontology (GO) terms or biological pathways related to metamorphosis were enriched in the tail compared to those in the intestine and hindlimb. This difference likely underlies the drastic effects of TRα knockout on the metamorphosis of the intestine and hindlimb but not the tail. Thus, TRα has tissue-specific roles in regulating T3-dependent anuran metamorphosis by directly targeting the pathways and GO terms important for metamorphosis.     

Molecular cloning and expression of TRα and TRβ in the protandrous cinnamon clownfish,Amphiprion melanopus during sex reversal

We divided the process of sex reversal from immature male to mature female in the protandrous cinnamon clownfish (Amphiprion melanopus) into six developmental stages as follows: I, immature male; II, mature male; III, male at 60 days after female removal; IV, male at 90 days after female removal; V, male at 120 days after female removal; and VI, mature female. Thyroid hormone receptors α (TRα) and β (TRβ) cDNAs were cloned from the ovary and mRNA expression levels were compared during the sex-reversal process. The nucleotide sequences of the TRα and TRβ cDNA were 1230 and 1188 bp in length with open reading frames encoding peptides of 409 and 395 amino acids, respectively. We observed that TRα mRNA and protein levels were high in all stages except the immature gonad, while TRβ mRNA levels were higher in the mature ovary than in any other gonadal stage. We injected gonadotropin-releasing hormone analogue to identify its effects on TRs mRNA in immature fish. The mRNA levels of TRs increased significantly. We therefore propose that TRs are related to testicular development as well as ovarian development in cinnamon clownfish. The present study also provides basic data on the role of TRs during sex reversal in fish.     

Growth and energy budget of F2'all-fish' growth hormone gene transgenic common carp

The growth and energy budget for F₂‘all‐fish’ growth hormone gene transgenic common carp Cyprinus carpio of two body sizes were investigated at 29·2° C for 21 days. Specific growth rate, feed intake, feed efficiency, digestibility coefficients of dry matter and protein, gross energy intake (I_E), and the proportion of I_E utilized for heat production (H_E) were significantly higher in the transgenics than in the controls. The proportion of I_E directed to waste products [faecal energy (F_E) and excretory energy loss (Z_E+U_E) where Z_E is through the gills and U_E through the kidney], and the proportion of metabolizable energy (M_E) for recovered energy (R_E) were significantly lower in the transgenics than in the controls. The average energy budget equation of transgenic fish was as follows: 100 I_E= 19·3 F_E+ 6·0 (Z_E+U_E) + 45·2 H_E+ 29·5 R_E or 100 M_E= 60·5 H_E+ 39·5 R_E. The average energy budget equation of the controls was: 100 I_E= 25·2 F_E+ 7·4 (Z_E+U_E) + 35·5 H_E+ 31·9 R_E or 100 M_E= 52·7 H_E+ 47·3 R_E. These findings indicate that the high growth rate of ‘all‐fish’ transgenic common carp relative to their non‐transgenic counterparts was due to their increased feed intake, reduced lose of waste productions and improved feed efficiency. The benefit of the increased energy intake by transgenic fish, however, was diminished by their increased metabolism.     

Effects of temperature and fish oil supplementation on ovarian development and foxl2 mRNA expression in spotted scat Scatophagus argus

In this study, the complete foxl2 complementary (c)DNA sequence was isolated by simple modular‐architecture research tool (SMART)er rapid amplification of cDNA ends (RACE). Two year‐old female spotted scat, Scatophagus argus, were reared at different temperatures (23, 26 and 29° C) for 6 weeks, or fed with different concentrations of dietary fish oil (0, 2 or 6%) for 8 weeks. Ovarian development, serum oestradiol‐17β (E₂) levels, as well as ovarian foxl2 expression were measured. At the end of experiment, ovarian foxl2 messenger (m)RNA expression in fish reared at 23 and 26° C was significantly higher than that in fish reared at 29° C, and that in 2 and 6% fish oil groups was also significantly higher than that in control group (P < 0·05). Serum E₂ levels exhibited the same trend with foxl2 mRNA expression in temperature treatment groups and fish oil fed groups. There was a significant positive correlation between stage of oocytes and foxl2 expressions. Results showed that from 23 to 29° C, the optimal temperature for ovarian development in S. argus was 23–26° C, and 6% fish oil supplementation could effectively promote ovarian development. Optimal temperature and fish oil supplement might increase ovarian foxl2 mRNA expressions to promote ovarian development in S. argus.     

Δ53β hydroxysteroid dehydrogenase activity of the rat corpus luteum exhibits positive substrate-binding co-operativity: A continuous monitoring microdensitometric study with unfixed ovarian tissue sections

Δ⁵3β hydroxysteroid dehydrogenase activity transforms biologically inactive Δ⁵3β hydroxy steroids into the active Δ⁴3-keto products (e.g. pregnenolone to progesterone). Using a cytochemical procedure which allows for the continuous microdensitometric monitoring of an enzyme reaction as it proceeds and a well described cytochemical assay for Δ⁵3β HSD we have analysed the initial velocity rates (V_o) for dehydroepiandrosterone (DHEA) binding to this enzyme in regressing (i.e. 20α hydroxy steroid dehydrogenase positive) corpus luteum (CL) cells in unfixed tissue sections (5 μm) of the dioestrous and proestrous rat ovary. The results are mean ± S.E.M. The relationship between DHEA concentration (0 to 50 μM) and Δ⁵3β HSD activity in the dioestrous corpora lutea was sigmoidal and had an atypical 1/V_o versus 1/S plot, the x intercept being positive. Using a 1/V_o versus 1/S² plot the V_max was determined to be 1·0 ± 0·08 μmol min^?1 mg^?1 CL (n = 6). The Hill constant was 2·7 ± 0·02 (n = 6) suggesting a high degree of positive co-operativity for DHEA binding. The S concentration for half maximal activity was 17 ± 1 μmoles (n = 6). In the corpora lutea cells of the proestrous ovary, the V_max for DHEA transformation was unchanged (0·95 ± 0·04 μmol min^?1 mg^?1, n = 3) whilst the S_0·5 was significantly increased to 27 ± 0·1 (p < 0·01, n = 3). The Hill constant remained positive being 2·9 ± 0·2 (n = 3). NAD⁺ binding to 3β HSD in regressing corpora lutea of the proestrous ovary has been demonstrated previously to be hyperbolic and fit the classical Michaelis-Menten model.¹ Extending the analysis of NAD⁺ binding to the regressing corpus luteum of the dioestrous rat ovary revealed similar kinetic characteristics to that seen with the proestrous enzyme, the apparent V_max and K_m being 0·84 ± 0·04 μmol min^?1 mg^?1 CL (n = 3) and 27 ± 7 μmol 1^?1 (n = 3) respectively. The Hill constant was 1·1 ± 0·03 (n = 3), indicating no co-operativity of co-factor binding.     

Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates release of somatolactin (SL)-α and SL-β from cultured goldfish pituitary cells via the PAC1 receptor-signaling pathway,and affects the expression of SL-α and SL-β mRNAs

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide that stimulates the release of adenohypophyseal hormone from the pituitary in fish. In the goldfish, PACAP induces the release of somatolactin (SL), in particular, from cultured pituitary cells. SL belongs to the growth hormone and prolactin family, and comprises two molecular variants termed SL-α and SL-β in goldfish. However, there is no information about the involvement of PACAP in the regulation of SL-α and SL-β release and the expression of their mRNAs. Therefore, we examined the effect of PACAP on SL-α and SL-β release from cultured goldfish pituitary cells. Treatment with PACAP (10⁻¹⁰–10⁻⁷ M) increased the release of both SL-α and SL-β. The stimulatory action of PACAP (10⁻⁹ M) on SL-α and SL-β release was blocked by treatment with a PACAP-selective receptor (PAC₁R) antagonist, PACAP_(6–38) (10⁻⁶ M). We also examined whether PACAP affects the expression of SL-α and SL-β mRNAs in cultured pituitary cells. Treatment with PACAP (10⁻⁹ and 10⁻⁸ M) for 6 h decreased the expression level of SL-α mRNA but increased that of SL-β mRNA. The action of PACAP (10⁻⁸ M) on SL-β mRNA expression was blocked by treatment with PACAP_(6–38) (10⁻⁶ M), whereas PACAP_(6–38) elicited no change in the expression of SL-α mRNA. These results indicate that in cultured goldfish pituitary cells, PACAP stimulates the release of SL-α and SL-β, and expression of SL-β mRNA, via the PAC₁R-signaling pathway. However, the mechanism whereby PACAP inhibits the expression of SL-α mRNA does not seem to be mediated by PAC₁R signaling.     

An energy budget for juvenile thick-lipped mullet, Crenimugil labrosus (Risso)

A series of experiments were carried out to construct an energy budget for juvenile thick lipped mullet, Crenimugil labrosus Risso. A partial factorial experimental design was used to examine the effects of temperature, fish size and meal size on growth. The maximum ration that the fish were able to ingest completely per day was found to be 0·8, 1·4 and 2·3% wet body weight (b.w.) at 13,18 and 23°C, respectively. Ingested maintenance requirements (M.R.) were estimated to be 137, 205 and 288 cal fish^-1 day^-1 at 13, 18 and 23°C, respectively. At 18deg; C, M.R. varied as 25 W^1.04 cal day^-1, where W= fish weight (g). Growth rate increased with increasing temperature. Maximal conversion efficiency was 21–24% and was achieved closer to the maximum ingested ration with increasing temperature. The relationship between respiration rate and W at 18deg; C for 3-20 g fish is described by: respiration rate (ml O₂ h^-1) = 0·128 W^0.976 The energy cost of apparent specific dynamic action at 18deg; C was found to vary between 5·1% and 23·6% of the calorific value of the ingested meal (1% wet b.w.) , mean (± S.E.)=10·2 ± 2·0%. Post mortem analyses of groups of fish fed 0·2, 0·8 and 1·5% wet b.w. meals showed a significant increase in total lipid and a significant decrease in water content with increasing ratio size. A negative correlation was found between body water content and total lipid (and calories). The mean assimilation efficiency (±s.e.) for 5–10 g mullet at 18deg; C was 73·9 ± 3·6%. The observations reported in this study were brought together to construct an energy budget for juvenile C. labrosus which was found to give a reliable prediction (within 10%) of energy demand and growth under the prevailing experimental conditions. Both gross (K₁) and net (K₂) growth efficiencies, based on energy values, increased with increasing ratio size up to satiation and were independent of temperature. The maximum values of K₁ and K₂ observed were 0·33 and 0·46, respectively. The third order efficiency (K₃) appeared to be independent of temperature and ration size; mean values ranged between 0·66 and 0·84.     

Effect of a pharmacological activation of PPAR on the expression of RAR and TR in rat liver

It has recently been shown that high-fat diets induce the expression of peroxisome proliferator-activated receptor (PPAR) with a concomitant decrease in expression of retinoic acid (RAR) and triiodothyronine (TR) receptors in rat liver. The authors have suggested that PPAR activation may be responsible for these modifications in nuclear receptor expression. With the aim of gaining further insight into a possible relationship between the patterns of expression of these receptors, we have examined, using a pharmacological model, the effect of a strong and specific PPAR activation induced by bezafibrate, a peroxisome proliferator agent. Activation of PPAR was evaluated by quantifying PPARα mRNA and acyl-CoA oxidase mRNA. The expression of RAR and TR was determined by assaying the binding properties of these nuclear receptors and by quantifying the mRNA level of RARβ and TRα₁,β₁ isoforms. After a 10 day treatment of young rats, induction of PPAR (PPARα mRNA was increased by 40% [P< 0.05 and acyl-CoA oxidase mRNA by 411% [P<0.001]) and a concomitant decrease of RAR and TR expression (Maximal Binding Capacity was decreased by 21 and 26%, respectively [P<0.05]) in the liver was observed. RXRα mRNA expression was unchanged by treatment. Cross-talk between RAR, TR and PPAR signalling pathways may be implicated in the new patterns of nuclear receptor expression observed. The decreased expression of RAR and TR reported here could provide a novel element for the understanding of the link between PPAR and tumorigenesis in rat liver.     

Di‐2‐ethylhexyl phthalate (DEHP) exposure disturbs lipid metabolism in juvenile yellow catfish Tachysurus fulvidraco

This study was conducted to determine the mechanism by which di‐2‐ethylhexyl phthalate (DEHP) exposure influences lipid metabolism of juvenile yellow catfish Tachysurus fulvidraco. Fish were exposed to three DEHP concentrations (0, 0·1 and 0·5 mg l^?1 DEHP) for 8 weeks. Fatty acid synthase (FAS) activity significantly decreased with increasing DEHP concentrations, the highest value was in the Tween control group, whereas the lowest activities of carnitine palmitoyltransferase (CPT) and lipoprotein lipase (LPL) were in this group. The messenger (m)RNA levels of 6‐phospho‐gluconate dehydrogenase (6PGD), FAS and acetyl‐CoA carboxylase a (ACCa) significantly increased with increasing DEHP concentration, the highest values were in the 0·5 mg l^?1 DEHP group. The mRNA level of peroxisome proliferator‐activated receptor γ (PPARγ) was lower in Tween control than in fish exposed to 0·1 and 0·5 mg l^?1 DEHP. The highest mRNA level of ACCb was in the 0·1 mg l^?1 DEHP group. These results indicate that DEHP exposure can disturb lipid metabolism at the enzymatic and mRNA levels in Pelteobagrus fulvidraco.     

THE ORIGIN OF CLOSED CIRCLES OF FIVE CHROMOSOMES IN OENOTHERA

Circles in Oenothera are normally composed of even numbers of chromosomes. Two cases of trisomic hybrids with closed circles containing an odd number of chromosomes are here reported. The first of these appeared to contain β Trois Pistoles I and ^hMateo and had a closed circle of 5, an open chain of 8, and a pair; or chains of 9 and 4 plus a pair. The second hybrid seemed to contain α Trois Pistoles II and β Palmer Lake and had a closed circle of 5 and an open chain of 10. It was possible to analyze in considerable detail the situation in the first of these. The chromosome 5·12 or 11·6 of or Trois Pistoles I was present in this trisomic, which arose either through the simple addition of 5·12 or 11·6 to the β Trois Pistoles I complement contributed through the egg, or through double non-disjunction. If it were a trisomic of the former type it contained only one chromosome of α Trois Pistoles I. If it arose through double non-disjunction, the minimum it could have had would be the equivalent of two α Trois Pistoles I chromosomes, and it might theoretically have had up to the equivalent of 5 chromosomes. It showed, however, no influence of α Trois Pistoles I in its phenotype, although the two Trois Pistoles I complexes produce very different phenotypes. It is probable, therefore, that the trisomic arose as a result of simple addition of 5·14 or 11·6 of α Trois Pistoles I to the β Trois Pistoles I complex received from the mother.     

Rapid action of estradiol in primate GnRH neurons: the role of estrogen receptor alpha and estrogen receptor beta

Estrogens play a pivotal role in the control of female reproductive function. Recent studies using primate GnRH neurons derived from embryonic nasal placode indicate that 17β-estradiol (E₂) causes a rapid stimulatory action. E₂ (1 nM) stimulates firing activity and intracellular calcium ([Ca²⁺]_i) oscillations of primate GnRH neurons within a few min. E₂ also stimulates GnRH release within 10 min. However, the classical estrogen receptors, ERα and ERβ, do not appear to play a role in E₂-induced [Ca²⁺]_i oscillations or GnRH release, as the estrogen receptor antagonist, ICI 182,780, failed to block these responses. Rather, this rapid E₂ action is, at least in part, mediated by a G-protein coupled receptor GPR30. In the present study we further investigate the role of ERα and ERβ in the rapid action of E₂ by knocking down cellular ERα and ERβ by transfection of GnRH neurons with specific siRNA for rhesus monkey ERα and ERβ. Results indicate that cellular knockdown of ERα and ERβ failed to block the E₂-induced changes in [Ca²⁺]_i oscillations. It is concluded that neither ERα nor ERβ is required for the rapid action of E₂ in primate GnRH neurons.