Plasticity of the prolactin (PRL) axis: mechanisms underlying regulation of output in female mice.

The output of prolactin (PRL) is highly dynamic with dramatic changes in its secretion from the anterior pituitary gland depending on prevailing physiological status. In adult female mice, there are three distinct phases of output and each of these is related to the functions of PRL at specific stages of reproduction. Recent studies of the changes in the regulation of PRL during its period of maximum output, lactation, have shown alterations at both the level of the anterior pituitary and hypothalamus. The PRL-secreting cells of the anterior pituitary are organised into a homotypic network in virgin animals, facilitating coordinated bouts of activity between interconnected PRL cells. During lactation, coordinated activity increases due to the changes in structural connectivity, and this drives large elevations in PRL secretion. Surprisingly, these changes in connectivity are maintained after weaning, despite reversion of PRL output to that of virgin animals, and result in an augmented output of hormone during a second lactation. At the level of the hypothalamus, tuberoinfundibular dopamine (TIDA) neurons, the major inhibitors of PRL secretion, have unexpectedly been shown to remain responsive to PRL during lactation. However, there is an uncoupling between TIDA neuron firing and dopamine secretion, with a potential switch to enkephalin release. Such a process may reinforce hormone secretion through dual disinhibition and stimulation of PRL cell activity. Thus, integration of signalling along the hypothalamo-pituitary axis is responsible for increased secretory output of PRL cells during lactation, as well as allowing the system to anticipate future demands.

Pituitary phenotypes of mice lacking the notch signalling ligand delta-like 1 homologue.

The Notch signalling pathway ligand delta-like 1 homologue (Dlk1, also named Pref1) is expressed throughout the developing pituitary and becomes restricted to mostly growth hormone (GH) cells within the adult gland. We have investigated the role of Dlk1 in pituitary development and function from late embryogenesis to adulthood using a mouse model completely lacking the expression of Dlk1. We confirm that Dlk1-null mice are shorter and weigh less than wild-type littermates from late gestation, at parturition and in adulthood. A loss of Dlk1 leads to significant reduction in GH content throughout life, whereas other pituitary hormones are reduced to varying degrees depending on sex and age. Both the size of the pituitary and the proportion of hormone-producing cell populations are unchanged, suggesting that there is a reduction in hormone content per cell. In vivo challenge of mutant and wild-type littermates with growth hormone-releasing hormone and growth hormone-releasing hexapeptide shows that reduced GH secretion is unlikely to account for the reduced growth of Dlk1 knockout animals. These data suggest that loss of Dlk1 gives rise to minor pituitary defects manifesting as an age- and sex-dependent reduction in pituitary hormone contents. However, Dlk1 expression in other tissue is most likely responsible for the weight and length differences observed in mutant animals.

Anterior pituitary cell networks.

Both endocrine and non-endocrine cells of the pituitary gland are organized into structural and functional networks which are formed during embryonic development but which may be modified throughout life. Structural mapping of the various endocrine cell types has highlighted the existence of distinct network motifs and relationships with the vasculature which may relate to temporal differences in their output. Functional characterization of the network activity of growth hormone and prolactin cells has revealed a role for cell organization in gene regulation, the plasticity of pituitary hormone output and remarkably the ability to memorize altered demand. As such, the description of these endocrine cell networks alters the concept of the pituitary from a gland which simply responds to external regulation to that of an oscillator which may memorize information and constantly adapt its coordinated networks' responses to the flow of hypothalamic inputs.

Two novel mutations in the POU1F1 gene generate null alleles through different mechanisms leading to combined pituitary hormone deficiency.

BACKGROUND: Mutations in the POU1F1 gene severely affect the development and function of the anterior pituitary gland and lead to combined pituitary hormone deficiency (CPHD). OBJECTIVE: The clinical and genetic analysis of a patient presenting with CPHD and functional characterization of identified mutations. PATIENT: We describe a male patient with extreme short stature, learning difficulties, anterior pituitary hypoplasia, secondary hypothyroidism and undetectable prolactin, growth hormone (GH) and insulin-like growth factor 1 (IGF1), with normal random cortisol. DESIGN: The POU1F1 coding region was amplified by PCR and sequenced; the functional consequence of the mutations was analysed by cell transfection and in vitro assays. RESULTS: Genetic analysis revealed compound heterozygosity for two novel putative loss of function mutations in POU1F1: a transition at position +3 of intron 1 [IVS1+3nt(A>G)] and a point mutation in exon 6 resulting in a substitution of arginine by tryptophan (R265W). Functional analysis revealed that IVS1+3nt(A>G) results in a reduction in the correctly spliced POU1F1 mRNA, which could be corrected by mutations of the +4, +5 and +6 nucleotides. Analysis of POU1F1(R265W) revealed complete loss of function resulting from severely reduced protein stability. CONCLUSIONS: Combined pituitary hormone deficiency in this patient is caused by loss of POU1F1 function by two novel mechanisms, namely aberrant splicing (IVS1+3nt (A>G) and protein instability (R265W). Identification of the genetic basis of CPHD enabled the cessation of hydrocortisone therapy without the need for further assessment for evolving endocrinopathy.

Continuous on-line monitoring of secretion from rodent pituitary endocrine cells using fluorescent protein surrogate markers.

We have developed a system to use secreted fluorescent proteins (FPs) as surrogate markers for the continuous on-line monitoring of hormone release from perfused tissue slices. We have tested this system using GH-GFP transgenic rats with green fluorescent protein (GFP) targeted to the secretory vesicles (SVs) of pituitary growth hormone (GH) cells. Brief exposures of vibratome slices to GH secretagogues [GH-releasing hormone (GHRH), GH-releasing peptide-6 (GHRP-6)] or somatostatin caused changes in FP output that correlate with hormone secretion, subsequently measured in fractions of perfusate by radioimmunoassay. The temporal resolution of this method was capable of revealing differences in the kinetics of response to GHRH and GHRP-6 between wild-type and dwarf (dw/dw) rats harbouring the GH-GFP transgene. We further tested the utility of the system by generating transgenic mice with red FPs targeted to secretory vesicles (PRL-mRFP(sv)) and to the cytoplasm (PRL-DsRed(cyto)) of lactotrophs. Dopamine had no effect on the FP output from pituitary slices of PRL-DsRed(cyto) mice but inhibited output from those of PRL-mRFP(sv) animals, with a rebound increase of release after removal, which again correlated with hormone output measured in the perfusate by radioimmunoassay. The inhibition of monomeric RFP secretion by dopamine was dose-dependent, as was stimulation by low concentrations of oxytocin. The temporal resolution afforded by this method provides useful insight into the release kinetics from large populations of pituitary cells, and fills a temporo-spatial gap between single vesicle and single cell monitoring of exocytosis in milliseconds, and in vivo sampling studies of release into the bloodstream on a time scale of minutes.

Host range and interference studies of three classes of pig endogenous retrovirus.

Recent interest in the use of porcine organs, tissues, and cells for xenotransplantation to humans has highlighted the need to characterize the properties of pig endogenous retroviruses (PERVs). Analysis of a variety of pig cells allowed us to isolate and identify three classes of infectious type C endogenous retrovirus (PERV-A, PERV-B, and PERV-C) which have distinct env genes but have highly homologous sequences in the rest of the genome. To study the properties of these env genes, expression plasmids for the three env genes were constructed and used to generate retrovirus vectors bearing corresponding Env proteins. Host range analyses by the vector transduction assay showed that PERV-A and PERV-B Envs have wider host ranges, including several human cell lines, compared with PERV-C Env, which infected only two pig cell lines and one human cell line. All PERVs could infect pig cells, indicating that the PERVs have a potential to replicate in pig transplants in immunosuppressed patients. Receptors for PERV-A and PERV-B were present on cells of some other species, including mink, rat, mouse, and dog, suggesting that such species may provide useful model systems to study infection and pathogenicity of PERV. In contrast, no vector transduction was observed on nonhuman primate cell lines, casting doubt on the utility of nonhuman primates as models for PERV zoonosis. Interference studies showed that the three PERV strains use receptors distinct from each other and from a number of other type C mammalian retroviruses.

Endogenous retroviruses: a potential problem for xenotransplantation?

To overcome the shortage of suitable human donors for transplantation attention has recently turned to the possibility of using genetically modified pigs as a source of cells and organs. It has been suggested that such procedures might facilitate the introduction of novel retroviruses, normally resident in the pig germ line, into the human population (Stoye and Coffin, Nature Medicine 1: 1100, 1995). The consequences of such a transfer remain unclear; however, the demonstration that certain porcine cell lines express infectious retroviruses which can infect human cells (Patience et al., Nature Medicine 3: 282-286, 1997) emphasizes that there are grounds for practical concern. We have now cloned the envelope genes of the expressed viruses and are using these clones in studies of the interaction of the porcine viruses with their cellular receptors. We have also initiated studies of the inheritance and expression of human-tropic endogenous proviruses present in different pig populations. These studies reveal that at least two classes of human-tropic endogenous porcine retrovirus are widely distributed in pigs (Le Tissier et al., Nature 389: 681-681, 1997). The implications of our results for assessing the potential risk of retroviral transfer during xenotransplantation are discussed.

The plasmacytoma resistance gene, Pctr2, delays the onset of tumorigenesis and resides in the telomeric region of chromosome 4.

Mouse plasmacytomas share pathogenetic features in common with both multiple myeloma and Burkitt's lymphoma in humans. Susceptibility to plasmacytoma induction by intraperitoneal pristane in mice is controlled by multiple genes. At least two of these genes reside on mouse chromosome 4 in regions of the genome sharing linkage homology with human chromosomes 9p21, 1p32, and 1p36. A series of congenic strains recombinant for regions of mouse chromosome 4 in the vicinity of the Pctr2 predisposition locus were created and typed for their tumor susceptibility/resistance phenotypes. These strains were derived by introgressively backcrossing alleles from resistant DBA/2 mice onto the susceptible BALB/cAnPt background. Six resistant and two susceptible strains were allelotyped for 10 genes and 49 random DNA markers to identify the smallest region of overlap in the resistant strains. These studies have determined that the Pctr2 locus resides in either a 500-kb interval proximal to Nppa, or in a 1- to 2-centiMorgan (cM) interval distal to Nppa. In these congenic strain analyses, the Nppa and Fv1 loci, in addition to genes within about 1 cM of these loci, have been excluded as candidates for the Pctr2 locus. A relevant locus that may reside in this interval is Rep2; it is associated with the efficiency of repairing X-ray induced DNA damage sustained during the G2 phase of the mitotic cycle. The Pctr2 locus acts in a codominant fashion. F1 hybrids between resistant and susceptible congenic strains exhibit a reduced tumor incidence and a significant delay in the onset of tumorigenesis. Identification and eventual cloning of the Pctr2 locus may assist in the identification of genes involved in many types of cancer showing aberrations in human chromosome 1p36.

Endogenous retroviruses and the evolution of resistance to retroviral infection.

The current AIDS epidemic has rekindled interest in the evolution of retroviruses and the development of resistance to infection. Retroviruses and their vertebrate hosts have coexisted for millions of years, during which time a variety of host defence mechanisms has evolved. One repeated strategy is to use endogenous retroviruses to combat infection by their exogenous relatives.

Positional cloning of the mouse retrovirus restriction gene Fv1.

Vertebrate evolution has taken place against a background of constant retrovirus infection, and much of the mammalian genome consists of endogenous retrovirus-like elements. Several host genes have evolved to control retrovirus replication, including Friend-virus-susceptibility-1, Fv1, on mouse chromosome 4 (refs 3, 4). The Fv1 gene acts on murine leukaemia virus at a stage after entry into the target cell but before integration and formation of the provirus. Although restriction is not absolute, Fv1 prevents or delays spontaneous or experimentally induced viral tumours. In vitro, Fv1 restriction leads to an apparent 50-1,000 fold reduction in viral titre. Genetic evidence implicates a direct interaction between the Fv1 gene product and a component of the viral preintegration complex, the capsid protein CA (refs 7-9). We have now cloned Fv1: the gene appears to be derived from the gag region of an endogenous retrovirus unrelated to murine leukaemia virus, implying that the Fv1 protein and its target may share functional similarities despite the absence of nucleotide-sequence homology.

Sequence analysis and genetic mapping of porcine chromosome 11 centromeric S0048 marker.

We report the existence of a new family of swine centromeric satellite DNA composed of a 51-bp repeat unit, most specifically found on pig chromosome 11 centromere and with less specificity at the centromeric region of other meta- and submetacentric chromosomes. This satellite DNA family, which has no homologies with the Mc1 and Ac2 families published previously, was named Mc2. We designed a specific primer set for PCR amplification of this centromeric satellite DNA. Specificity of amplification was checked by using a porcine somatic cell hybrid panel and by FISH. Furthermore, the development of a PCR-RFLP marker of Mc2 repetition allowed its genetic mapping on the PiGMaP reference families panel. The centromere of chromosome 11 was thus integrated to the genetic map previously published.

Development of an assay method for purine catabolic enzymes in the mouse and its adaptation for use on an autoanalyzer.

An assay method has been developed for the purine catabolic enzymes adenosylhomocysteinase, adenosine deaminase (ADA), purine-nucleoside phosphorylase (PNP), and urate oxidase in mice. The assay links H2O2 produced during purine catabolism to the production of a dye complex. The assay method has been developed for ADA and PNP in erythrocytes and for all four enzymes in liver. The assay is cheap, sensitive, and easy to perform. The dye complex absorbs in the visible range, negating the need for an expensive ultraviolet spectrophotometer and allowing the use of an autoanalyzer.

Rapid mapping of cosmid clones on pig chromosomes by fluorescence in situ hybridization.

Nineteen cosmids have been mapped to pig chromosomes by fluorescence in situ hybridization. Two kinds of cosmid clones were isolated as potential physical and genetic markers for the pig genome. Anonymous cosmids were obtained by screening a commercial cosmid library and were localized to Chromosomes (Chrs) 1, 2, 6, 7, 8, 10, 11, 12, 13, and 14. Some of these cosmids were found to reveal RFLP type DNA polymorphism. Microsatellite-containing cosmid clones were isolated by screening a pig cosmid library with a (CA)10 probe and were regionally mapped to Chrs 2, 6, 7, 13, and 14. Ten of the 19 chromosomes in the pig were labeled with these probes. Two-color fluorescence in situ hybridization was used to increase the efficiency of the cosmid localizations.

Mapping of the structural gene for S-adenosyl homocysteine hydrolase to mouse chromosome 2, and related sequences to chromosomes 8 and X.

Comparative mapping studies in human and mouse have shown that, to date, human Chromosome (Chr) 20 is completely syntenic with distal mouse Chr 2. The structural locus for S-adenosyl-L-homocysteine hydrolase (EC 3.3.1.1) in human, AHCY, maps to 20 qter-->q13.1, and we report here that the homologous locus in the mouse, Ahcy, maps to distal mouse Chr 2 with gene order Pcna-Ahcy-Ada. Analysis of 123 progeny of an interspecific backcross between a laboratory stock, AN, and Mus spretus using a rat cDNA probe revealed the presence of at least two other Ahcy-related sequences segregating independently in the mouse genome. One, Ahcy-rs1, was mapped to Chr 8 in the BXH recombinant inbred strains, and the other, Ahcy-rs2, shows a pattern of inheritance consistent with X-linkage.