No abstract

The sex-linked dwarf (SLD) chicken, which lacks GH receptor (GHR), and its normal littermates provide a useful experimental system to investigate GH-dependent cellular responses. The GH dependence of insulin-like growth factor I (IGF-I) expression in tissues was examined in SLD and normal chickens of the Gifu 20 strain. Four weeks after hatching, the most abundant expression of IGF-I messenger RNA (mRNA) was observed in liver of normal chickens, whereas no IGF-mRNA expression was detected in that organ of dwarf chickens. On the contrary, in extrahepatic tissues such as spleen, lung, brain, kidney, heart, intestine, thymus, and muscle, IGF-I mRNA expression was equally observed in normal and GHR-lacking dwarf chickens. In the testis, expression of IGF-I mRNA was enhanced by about 5-fold in dwarf chickens showing an expression level comparable to that in normal liver. On day 16 in the embryonic stage, IGF-I mRNA was expressed in muscle, brain, eye, heart, and lung in both normal and SLD chick embryos. However, no IGF-I mRNA expression was observed in liver or kidney of normal and dwarf chick embryos. These results suggest that in chicken, IGF-I mRNA is expressed in liver in a GH-dependent manner after hatching, whereas in other tissues, mRNA expression is independent of GH and GHR before and after hatching, except for testis, in which GH seems to inhibit IGF-I mRNA expression.

Prolactin receptor (PRL-R) mRNA expression levels in the female rat brain (cerebrum) during pup contact stimulation were determined by the reverse transcription-PCR method. The high expression levels of long form PRL-R mRNA found in the brain of lactating rats were markedly reduced by removal of pups, and long form PRL-R mRNA levels were recovered by resumption of pup contact. Interestingly, pup contact stimuli of nulliparous virgin rats also markedly induced long form but not short form PRL-R mRNA expression in the brain in 1.3 days, together with the expression of maternal behaviour. In ovariectomized (OVX) or hypophysectomized (HYPOX) virgin rats, or in OVX plus HYPOX virgin rats, however, brain long form PRL-R mRNA was not significantly induced by pup contact stimuli for as long as 7 days, while maternal behaviour was fully expressed in these rats after 7 days of pup contact. The in situ hybridization experiments revealed that the long form PRL-R mRNA induced in virgin rats in contact with pups or in lactating rats was localized in the epithelial cells of the choroid plexus. No significant increase in mRNA was detected in other regions of the brain, such as the hypothalamus or cortex, in these maternal female rats. These results suggest that pup contact induces the expression of long form PRL-R mRNA in the choroid plexus of the brain in the presence of female sex steroid and pituitary hormones for the rapid expression of maternal behaviour. Our studies also suggested that maternal behaviour can be expressed in OVX or HYPOX rats after exposure to pups for 7 days without any significant increase in brain PRL-R mRNA expression.

Hormonal control of brain functions is considered to be important in the tolerance of stress, and it is now established that stress elevates serum PRL levels in male or cycling female rats. To investigate whether or how serum PRL acts on the brain during exposure to stress, we analyzed serum PRL levels and the gene expression of brain PRL receptors in rats subjected to restraint stress in the water (RSW). The serum PRL concentration was remarkably increased within 30 min in the rats by exposure to RSW and decreased to the initial level after 4 h of RSW, remaining at this level for up to 7 h of RSW. After the rats were released from the stress, the serum PRL level was significantly lowered in 6 h. Ribonuclease protection assay and in situ hybridization analysis revealed that messenger RNA (mRNA) expression for the long form PRL receptor [PRL-R(L)] was remarkably induced in the rat choroid plexus in 2 h of RSW. The high expression level of PRL-R(L) mRNA in the region was reduced after the rats were released from the stress. PRL-R(L) mRNA expression in the hypothalamus was at lower levels than those in the choroid plexus before and during the RSW treatment. The short form PRL receptor mRNA expression in the rat brain was considerably lower than expression of the long form receptor mRNA before or during RSW. The results indicated that the restraint stress caused a rapid increase in serum PRL and induced the gene expression for PRL-R(L) in the choroid plexus, suggesting stress-induced and choroid plexus PRL-R(L)-mediated transport of serum PRL into the cerebrospinal fluid.

No abstract

Prolactin (PRL) exerts a wide variety of physiological effects on mammalian tissues through its receptor (PRL-R) on the target cells. PRL-R in rat tissue consists of two isoforms, the long and the short form, and the regulatory mechanisms of their mRNA expression in tissues are complex and diverse. The present study reports the differential regulation of PRL-R mRNA expression in rat liver and kidney by testosterone and oestradiol. Using Northern blot analysis, short form PRL-R mRNA was clearly detected in female rat liver and male rat kidney, and long form PRL-R mRNA was faintly observed only in female rat liver. However, the reverse transcription-polymerase chain reaction method enabled efficient analysis of mRNA levels in short and long forms of PRL-R in the liver and kidney of both male and female rats. The mRNA levels for the long and short forms of PRL-R were depressed in the liver of male rats but not in that from female rats during sexual maturation. Castration of male rats resulted in the induction of the mRNAs for these two forms of PRL-R in the liver. Testosterone, but not oestradiol, completely blocked the induction by castration of liver PRL-R gene expression. In kidney, in contrast, mRNA levels for both forms of PRL-R were depressed in female rats but not in male rats after sexual maturation. Administration of oestradiol, but not of testosterone, caused marked repression of short form PRL-R mRNA, particularly in the kidney of male rats. The levels of long form PRL-R mRNA in the kidney was less affected by the administration of oestradiol. These results have suggested that the expression of PRL-R mRNAs in rat liver and kidney is differentially regulated by testosterone and oestrogen.

Prolactin (PRL) is considered to induce maternal behavior toward foster young in female rats. In the present study, we studied the relationship between pup contact-induced maternal behavior and serum PRL concentrations and brain PRL receptor (PRL-R) mRNA expression in male rats. Both intact and castrated male rats exposed to foster pups gradually developed caretaking behavior such as crouching and licking, but their exhibitions of other maternal behavior components, retrieval/grouping and nest building, were incomplete. However, in the male rats displaying crouching and licking, the concomitant increases in serum PRL concentration and brain mRNA expression for long-form PRL-R were observed. The expression of short-form PRL-R mRNA in the brain was not stimulated by pup contact. Administration of PRL remarkably promoted the onset of those maternal responses in male rats. On the other hand, when an intact male rat was housed in a cage where a lactating female rat and her pups were living, his scores in maternal behavior tests toward pups were lowered. And, concomitantly, increases in serum PRL concentration and brain expression of long-form PRL-R mRNA were reduced. In castrated male rats, however, the ratings of maternal behavior toward foster young, serum PRL concentration increase, or brain long-form PRL-R mRNA expression were not reduced at all by cohabitation with a female and her pups. These findings indicated that maternal behavior was triggered and maintained in pup-contacted male rats through elevated serum PRL levels and induced brain long-form PRL-R.

To understand the comprehensive mechanisms of gene expression and processing for insulin-like growth factor-I (IGF-I) in vertebrates, we have investigated the gene organization, promoter and transcriptional initiation sites, alternative splicing and polyadenylating sites, and the cDNA structures of this gene in the Japanese flounder, Paralichthys olivaceus. The flounder IGF-I gene was found to be composed of five exons and four introns spanning 17.5 kb. By Northern blot analysis, two major mRNA classes of 4.7 kb and 2.9 kb were found in the liver. cDNA cloning and reverse transcription polymerase chain reaction (RT-PCR) analysis indicated that these two mRNA classes result from two different-sized 3'-noncoding regions generated by alternative usage of two polyadenylating signals. Further analysis by RT-PCR and sequencing revealed that these mRNA classes both contain two subclasses of mRNA encoding two forms of IGF-I prepropeptide, preproIGF-I-1 and preproIGF-I-2. The two forms of preproIGF-I share the identical signal peptide and mature IGF-I domain but contain different E domains as a result of alternative splicing in exon 3. The mature form of flounder IGF-I was found to comprise 68 amino acid residues, showing a small molecular weight, 7486. In the 5'-flanking region, one major and four minor transcription start sites have been identified by ribonuclease protection assay between -230 and -130 from the translation initiation codon, but no canonical TATA box or GC box was detected in their upstream regions up to -724. The results suggest that some unknown transcription initiation factors are functioning in the promotion of IGF-I gene expression.