The role of prolactin and growth hormone in mammary gland development

Development and differentiation of the mammary gland occur primarily during pregnancy. Females homozygous (-/-) for the null mutation of the PRL receptor (PRLR) gene are sterile due to a complete failure of blastocysts to implant. In progesterone-treated mice pregnancy is rescued but the mammary gland is severely underdeveloped. Interestingly, females hemizygous for the PRLR (+/-) in their first lactation show an almost complete failure to lactate. This phenotype disappears in the second and subsequent pregnancies in inbred 129/Sv mice but is maintained in inbred C57BL/6 mice. In GH receptor (GHR) KO mice litter size is markedly decreased, probably due to an ovarian defect. To assess the relevance of the GH and PRLRs in the mammary gland development, GHR and PRLR null epithelia were transplanted into cleared fat pads of wild-type mice. Such studies show that epithelial GHR is not required for functional mammary development. In contrast, epithelial PRLRs are required for mammary development and milk protein gene expression during pregnancy. Since ductal development is impaired in GHR -/- mice, it appears that GH signals through the stromal compartment. In summary, it is now established that GH and PRL activate Stat5 in separate compartments, reflecting their specific roles in ductal and alveolar development and differentiation.     

Daily energy balance in growth hormone receptor/binding protein (GHR −/−) gene-disrupted mice is achieved through an increase in dark-phase energy efficiency

The goal of this study was to examine factors that contribute to energy balance in female GHR ?/? mice. We measured energy intake, energy expenditure (EE), fuel utilization, body mass (M_b) changes and physical activity in 17 month-old female GHR ?/? mice and their age-matched wild type littermates. The GHR ?/? mice were smaller, consumed more food per unit M_b, had greater EE per unit M_b and had an increase in 24-h EE/M_b that was similar to the increase in their surface-area-to-volume ratio. Locomotor activity (LMA) was reduced in the GHR ?/? mice, but the energetic cost associated with their LMA was greater than in wild type controls. Furthermore, M_b and LMA were independent explanatory covariates of most of the variance in EE, and when adjusted for M_b and LMA, the GHR ?/? mice had higher EE during both the light and dark phases of the daily cycle. Respiratory quotient was lower in GHR ?/? mice during the light phase, which indicated a greater utilization of lipid relative to carbohydrate in these mice. Additionally, GHR ?/? mice had higher ratios of caloric intake to EE at several intervals during the dark phase, and this effect was greater and more sustained in the final 3 h of the dark phase. Therefore, we conclude that GHR ?/? mice are able to overcome the substantial energetic challenges of dwarfism through several mechanisms that promote stable M_b. Relative to wild type mice, the GHR ?/? mice consumed more calories per unit M_b, which offset the disproportionate increase in their daily energy expenditure. While GHR ?/? mice oxidized a greater proportion of lipid during the light phase in order to meet their energy requirements, they achieved greater energy efficiency and storage during the dark phase through a combination of higher energy consumption and lower LMA.     

The major histocompatibility complex region marked by HSP70-1 and HSP70- 2 variants is associated with clozapine-induced agranulocytosis in two different ethnic groups

Genes of the major histocompatibility complex (MHC) have been associated with susceptibility to drug-induced adverse reactions. We previously found that clozapine-induced agranulocytosis (CA) is associated with the HLA-DRB1*0402, DRB4*0101, DQB1*0302, DQA1*0301 haplotype in Ashkenazi Jewish patients and with the HLA-DRB1*1601, DRB5*02, DQB1*0502, DQA1*0102 haplotype in non-Jewish patients. In the present study, we tested the hypothesis that the variants of the heat- shock protein 70 (HSP-70) encoded by the HSP-70 loci located within the MHC region and known to be involved in apoptosis and regulation of cell proliferation could play an important role in molecular mechanisms of CA. First, we analyzed HSP70-2 polymorphism in risk-associated haplotypes from HLA homozygous cells and normal individuals and confirmed that the HSP70-2 9-kb variant was associated invariably with DR4 (HLA-DRB1*0402, DQB1*0302) and DR2 (HLA-DRB1*01601, DQB1*0502, DQA1*0102 and HLA-DRB1*1501, DQB1*0602) haplotypes, which were the haplotypes found increased in Jewish and non-Jewish patients with CA, respectively. The 9.0-kb variant was also found to be associated with HLA-B44, DRB1*0401 and HLA-B44, DRB1*07 haplotypes. Second, in patients with CA (12 Ashkenazi Jewish and 20 non-Jewish patients), HSP70-1 A and HSP70-2 9.0-kb variants were associated with the MHC haplotypes found by us to be markers of susceptibility to CA. The clozapine-treated control group had an excess number of HSP70-1 C and HSP70-2 8.5-kb variants, consistent with genetic resistance to CA associated with those variants. This finding supports our hypothesis that a dominant gene within the MHC region (marked by HSP70-1 and HSP70-2), but not necessarily HLA, is associated with CA in two different ethnic groups.     

Colonoscopic yield of colorectal neoplasia in daily clinical practice

AIM： To assess the prevalence and location of advanced neoplasia in patients undergoing colonoscopy, and to compare the yield per indication. METHODS： In a multicenter colonoscopy survey （n = 18 hospitals） in the Amsterdam area （Northern Holland）, data of all colonoscopies performed during a three month period in 2005 were analyzed. The location and the histological features of all colonic neoplasia were recorded. The prevalence and the distribution of advanced colorectal neoplasia and differences in yield between indication clusters were evaluated. Advanced neoplasm was defined as adenoma 〉 10 mm in size, with 〉 25% villous features or with high-grade dysplasia or cancer. RESULTS： A total of 4623 eligible patients underwent a total colonoscopy. The prevalence of advanced neoplasia was 13%, with 281 （6%） adenocarcinomas and 342 （7%） advanced adenomas. Sixty-seven percent and 33% of advanced neoplasia were located in the distal and proximal colon, respectively. Of all patients with right-sided advanced neoplasia （n = 228）, 51% had a normal distal colon, whereas 27% had a synchronous distal adenoma. Ten percent of all colonoscopies were performed in asymptomatic patients, 7% of whom had advanced neoplasia. In the respective procedure indication clusters, the prevalence of rightsided advanced neoplasia ranged from 11%-57%. CONCLUSION： One out of every 7-8 colonoscopies yielded an advanced colorectal neoplasm. Colonoscopy is warranted for the evaluation of both symptomatic and asymptomatic patients.     

Testicular endocrine function in GH receptor gene disrupted mice.

The consequences of disruption of GH receptor gene in GH receptor knockout mice on testicular function were evaluated. Adult male GH receptor knockout mice and their normal siblings were divided in to two subgroups and treated with either saline or ovine LH (0.3 microg/g BW) in saline. One hour after saline or LH administration, blood was obtained via heart puncture. Plasma IGF-I, LH, FSH, PRL, androstenedione, and testosterone levels were measured by RIAs. Testicular LH and PRL receptor numbers as well as pituitary LHbeta-subunit and testicular sulfated glycoprotein-2 mRNA levels were measured. Also, testicular morphometric analysis was performed. Unlike in normal, wild-type mice, the circulating IGF-I was undetectable in GH receptor knockout mice. The plasma PRL levels were (P<0.01) higher in GH receptor knockout mice than in their normal siblings. The basal LH secretion was similar in normal and GH receptor knockout mice. However, the circulating FSH levels were lower (P<0.001) in GH receptor gene disrupted mice. Administration of LH resulted in a significant (P<0.001) increase in plasma testosterone levels in both GH receptor knockout and normal mice. However, this testosterone response was attenuated (P < 0.01) in GH receptor knockout mice. Plasma androstenedione responses were similar in both GH receptor knockout and normal mice. Testicular LH and PRL receptor numbers were significantly decreased in GH receptor knockout mice. The results of the morphometric analysis of the testis revealed that the Leydig cell volume per testis was reduced in mice with GH receptor gene disruption. The steady-state of LHbeta-subunit and testicular sulfated glycoprotein-2 mRNA levels were not different in GH receptor knockout mice relative to their normal siblings. The present in vivo study demonstrates that in GH receptor knockout mice, LH action on the testis in terms of testosterone secretion is significantly attenuated and suggests that this is due to a decrease in the number of testicular LH receptors. The reduced number of PRL receptors may contribute to the diminished responsiveness of testicular steroidogenesis to LH by decreased ability to convert androstenedione to testosterone. These changes are most likely due to the absence of circulating IGF-I. These findings provide evidence that systemic IGF-I plays a major modulatory role in testicular endocrine function.     

The molecular basis of renal amyloidosis in Irish-American and Polish- Canadian kindreds

Hereditary amyloidosis of an unusual form has been reported in two separate kindreds; one was Polish-Canadian and the other was Irish- American (Am J Med 1975; 59:121 and Trans Assoc Am Physicians 1981; 94:211). In both kindreds, affected members developed hypertension and nephrotic syndrome due to amyloidosis in their forties or fifties, but the genetic background responsible for the condition has been left undetermined. To identify the genetic defect in these kindreds, a portion of exon 5 of the fibrinogen alpha-chain gene in members of these kindreds was examined for a mutation by single-strand conformation polymorphism analysis and direct DNA sequencing. DNA analyses revealed an A-->T transversion at the second base of codon 526 of the fibrinogen alpha-chain gene in both of these kindreds. Analysis of DNA polymorphisms in the fibrinogen alpha-chain gene locus (TCTT repeat in intron 3, Rsal site in exon 5, and Taql site in the 3' flanking region of the gene) showed the haplotype B5-Rsal(+)-Taql(-) for the Val 526 mutant gene in both kindreds studied here, as well as in two kindreds previously described (J Clin Invest 1994; 93:731). The fibrinogen alpha-chain gene mutation (Val 526) is the genetic defect responsible for hereditary renal amyloidosis in these two kindreds, and the mutant genes in the Val 526 kindreds may have been derived from a single founder.      

Developmental aspects of adipose tissue in GH receptor and prolactin receptor gene disrupted mice: site-specific effects upon proliferation, differentiation and hormone sensitivity

Direct metabolic effects of GH on adipose tissue are well established, but effects of prolactin (PRL) have been more controversial. Recent studies have demonstrated PRL receptors on adipocytes and effects of PRL on adipose tissue in vitro. The role of GH in adipocyte proliferation and differentiation is also controversial, since GH stimulates adipocyte differentiation in cell lines, whereas it stimulates proliferation but inhibits differentiation of adipocytes in primary cell culture. Using female gene disrupted (ko) mice, we showed that absence of PRL receptors (PRLRko) impaired development of both internal and s.c. adipose tissue, due to reduced numbers of adipocytes, an effect differing from that of reduced food intake, where cell volume is decreased. In contrast, GHRko mice exhibited major decreases in the number of internal adipocytes, whereas s.c. adipocyte numbers were increased, even though body weight was decreased by 40-50%. The changes in adipose tissue in PRLRko mice appeared to be entirely due to extrinsic factors since preadipocytes proliferated and differentiated in similar fashion to wild-type animals in vitro and their response to insulin and isoproterenol was similar to wild-type animals. This contrasted with GHRko mice, where s.c. adipocytes proliferated, differentiated, and responded to hormones in identical fashion to controls, whereas parametrial adipocytes exhibited markedly depressed proliferation and differentiation potential and failed to respond to insulin or noradrenaline. Our results provide in vivo evidence that both GH and PRL stimulate differentiation of adipocytes but that the effects of GH are site specific and induce intrinsic changes in the precursor population, which are retained in vitro.     

The growth hormone (GH)-axis of GH receptor/binding protein gene-disrupted and metallothionein-human GH-releasing hormone transgenic mice: hypothalamic neuropeptide and pituitary receptor expression in the absence and presence of GH feedback

Elevation of circulating GH acts to feed back at the level of the hypothalamus to decrease GH-releasing hormone (GHRH) and increase somatostatin (SRIF) production. In the rat, GH-induced changes in GHRH and SRIF expression are associated with changes in pituitary GHRH receptor (GHRH-R), GH secretagogue receptor (GHS-R), and SRIF receptor subtype messenger RNA (mRNA) levels. These observations suggest that GH regulates its own synthesis and release not only by altering expression of key hypothalamic neuropeptides but also by modulating the sensitivity of the pituitary to hypothalamic input, by regulating pituitary receptor synthesis. To further explore this possibility, we examined the relationship between the expression of hypothalamic neuropeptides [GHRH, SRIF, and neuropeptide Y (NPY)] and pituitary receptors [GHRH-R, GHS-R, and SRIF receptor subtypes (sst2 and sst5)] in two mouse strains with alterations in the GH-axis; the GH receptor/binding protein gene-disrupted mouse (GHR/BP-/-) and the metallothionein promoter driven human GHRH (MT-hGHRH) transgenic mouse. In GHR/BP-/- mice, serum insulin-like growth factor I levels are low, and circulating GH is elevated because of the lack of GH negative feedback. Hypothalamic GHRH mRNA levels in GHR/BP-/- mice were 232 +/- 20% of GHR/BP+/+ littermates (P < 0.01), whereas SRIF and NPY mRNA levels were reduced to 86 +/- 2% and 52 +/- 3% of controls, respectively (P < 0.05; ribonuclease protection assay). Pituitary GHRH-R and GHS-R mRNA levels of GHR/BP-/- mice were elevated to 275 +/- 55% and 319 +/- 68% of GHR/BP+/+ values (P < 0.05, respectively), whereas the sst2 and sst5 mRNA levels did not differ from GHR/BP intact controls as determined by multiplex RT-PCR. Therefore, in the absence of GH negative feedback, both hypothalamic and pituitary expression is altered to favor stimulation of GH synthesis and release. In MT-hGHRH mice, ectopic hGHRH transgene expression elevates circulating GH and insulin-like growth factor I. In this model of GH excess, endogenous (mouse) hypothalamic GHRH mRNA levels were reduced to 69 +/- 6% of nontransgenic controls, whereas SRIF mRNA levels were increased to 128 +/- 6% (P < 0.01). NPY mRNA levels were not significantly affected by hGHRH transgene expression. Also, MT-hGHRH pituitary GHRH-R and GHS-R mRNA levels did not differ from controls. However, sst2 and sst5 mRNA levels in MT-hGHRH mice were increased to 147 +/- 18% and 143 +/- 16% of normal values, respectively (P < 0.05). Therefore, in the presence of GH negative feedback, both hypothalamic and pituitary expression is altered to favor suppression of GH synthesis and release.     

Tissue-specific regulation of growth hormone (GH) receptor and insulin-like growth factor-I gene expression in the pituitary and liver of GH-deficient (lit/lit) mice and transgenic mice that overexpress bovine GH (bGH) or a bGH antagonist

GH has diverse biological actions that are mediated by binding to a specific, high-affinity cell surface receptor (GHR). Expression of GHR is tissue specific and a requirement for cellular responsiveness to GH. IGF-I is produced in multiple tissues and regulated in part by GH through GHR. In this study, we evaluated GHR and IGF-I mRNA expression in pituitary gland and compared the levels with those derived from liver of bovine GH transgenic, GH antagonist transgenic, lit/lit mice, and their respective controls using real-time RT-PCR. In liver, both GHR and IGF-I mRNA expressions were regulated in parallel with GH action in all three animal models, and there was a strong correlation between GHR and IGF-I mRNA levels. In the pituitary gland, increased expression of IGF-I mRNA in the pituitary of bovine GH transgenic mice was observed, whereas IGF-I expression in GH antagonist transgenic or lit/lit mice was similar to that observed in control animals. There were no differences of GHR mRNA levels in pituitary gland of any groups we examined. There was also no correlation between GHR and IGF-I mRNA levels in any group in the pituitary gland. In conclusion, we found that hepatic GHR and IGF-I mRNA levels were strongly correlated with each other in chronic GH excess or deficient state, and that regulation and correlation between local GHR and IGF-I mRNA levels induced by GH is different between liver and pituitary gland.     

Gamma (immune) interferon production by leukocytes from a patient with a TG cell proliferative disease

We report a patient with a disease characterized by proliferation of T cells with Fc receptors for IgG (TG). However, unlike lymphoid cells from normal individuals or from patients with other lymphoid malignancies, the patient's lymphocytes spontaneously produced gamma interferon (IFN-gamma) in vitro. The peripheral lymphocytes consisted of 95% TG cells, which exhibited the morphological characteristics of T- cell chronic lymphocytic leukemia (CLL) and were normal on cytochemical and chromosome analysis. The majority of TG cells were OKT3+, OKT8+, and OKT4-, 3A1-. These cells failed to express suppressor cell activity and displayed depressed levels of natural killer activity, but mediated antibody-dependent cell-mediated cytotoxicity. The spontaneous production of IFN-gamma by human peripheral lymphoid cells as demonstrated in this study may serve as a probe for studying the relationship between IFN-gamma and the proliferation of human T-cell subsets.