Dwarf mice and the ageing process.
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Aging↗
Biomedical subjects
A Bartke
Publications and source records attributed to A Bartke.
Increased plasma corticosterone levels in bovine growth hormone (bGH) transgenic mice: effects of ACTH, GH and IGF-I on in vitro adrenal corticosterone production.
Previous work from our laboratory provided evidence for increased plasma corticosterone levels in mice transgenic for human and bovine growth hormone (GH). Corticosterone was elevated in both sexes, under both basal and ether-induced stress conditions. The objectives of the present study were to investigate the in vitro adrenal sensitivity to ACTH, GH and/or IGF-I in normal and bGH transgenic mice, to examine plasma corticosterone levels at different times of the day, and to determine plasma levels of ACTH in these animals. For the measurement of plasma corticosterone and ACTH levels, transgenic and normal siblings were housed 2 per cage and decapitated simultaneously within 20 seconds of the first disturbance of the cage. The corticosterone production by in vitro adrenal incubations did not differ between adrenals from normal and transgenic mice at the basal level or in the presence of different doses of ACTH. Growth hormone or IGF-I did not have any effect on corticosterone production in vitro when given alone, and did not modify the effects of ACTH on the accumulation of corticosterone production in vitro when alone, and did not modify the effects of ACTH on the accumulation of corticosterone in the media. Plasma corticosterone concentrations were higher in transgenic than in normal animals in both morning and evening. Plasma concentrations of ACTH in animals killed in the morning were sharply increased in transgenic males as compared with their normal siblings. The results indicate that increased circulating levels of corticosterone in transgenic mice are not due to a potentiation of ACTH actions by GH or IGF-I, but rather to a chronic increase in plasma ACTH levels. The increase in ACTH is presumably a reflection of GH actions in the hypothalamic-pituitary system.
Adrenal Cortex↗
Overexpression of bovine growth hormone in transgenic mice is associated with changes in hepatic insulin receptors and in their kinase activity.
Transgenic mice expressing a hybrid gene produced by linking the promoter regulatory region of phosphoenolpyruvate carboxykinase (PEPCK) gene to the bovine growth hormone (bGH) gene, were used to investigate the effects of GH on insulin binding and insulin dependent tyrosine kinase activity of hepatic insulin receptors. Transgenic mice had normal levels of blood glucose, despite hyperinsulinemia, indicating that these animals were insulin resistant. The number of insulin receptors in the liver of transgenic mice was significantly decreased in both the particulate fraction (25%) and the solubilized membranes (40%) indicating that expressed (functional) and non-expressed (cryptic) receptors were affected. Scatchard analysis of competitive binding curves for insulin indicated that the affinity of the receptor did not differ between transgenic and normal mice. Insulin dependent tyrosine kinase activity in insulin receptors partially purified by wheat germ agglutin (WGA) agarose chromatography from solubilized liver membranes, was measured. The stimulatory action of insulin on phosphorylation of the synthetic substrate (a copolymer Glu-Tyr, 4:1) was increased 100% in transgenic, as compared to normal mice, using the same binding activity. Since transgenic mice are hyperinsulinemic, it is likely that the decreased insulin binding in this group reflects down regulation of the expressed and non-expressed insulin receptors, and the increased kinase activity represents a compensatory mechanism. We conclude that alterations in the insulin receptor number and in the tyrosine kinase activity develop in response to changes in insulin levels. Thus, insulin resistance detected in the liver of transgenic mice overexpressing GH may be due to post receptor defects.
Animals↗
Development of a specific radioimmunoassay for neuropeptide K: its application to Siberian hamster tissues.
In this report we describe the development of a radioimmunoassay for neuropeptide K (NPK) that is not influenced by the presence of free neurokinin A (NKA) or neuropeptide gamma (NPG). The antisera for the radioimmunoassay were obtained by immunizing rabbits with beta-preprotachykinin A fragment 69-91. This fragment contains the sequence 1-20 of NPK and has only the sequence 18-20 (Gly-His-Gly) in common with NPG. Two antisera were obtained, neither cross-reacting with NKA or NPG. The specificity of these antisera was confirmed by the finding of a major immunoreactive peak in the eluates obtained from HPLC separation, corresponding to NPK. This radioimmunoassay was applied to the determination of NPK in hypothalamic and anterior pituitary extracts from Siberian hamsters. Hypothalami from male and female hamsters contained similar concentrations of NPK, although slightly higher in males. NPK levels in the hypothalamus of female Siberian hamsters showed few fluctuations during the estrous cycle, with the levels measured at estrus being significantly lower than at proestrus, diestrus I, and diestrus II. In anterior pituitaries from Siberian hamsters the concentrations of NPK were significantly lower in females than in males. This radioimmunoassay has demonstrated the presence of NPK in the anterior pituitary gland of the Siberian hamster, and it should be very useful for future studies on mechanisms of regulation of NPK secretion in different tissues.
Amino Acid Sequence↗
Effects of photoperiod on hypothalamic and anterior pituitary tachykinins of male Siberian hamsters during development.
In previous reports from our laboratory we showed that the hypothalamus and especially the anterior pituitary gland of the Siberian hamster contain very high concentrations of substance P- and neurokinin A-like immunoreactive substances, as compared with other common laboratory rodents. It was thought, therefore, that a study of the developmental changes of these two tachykinins in the hypothalamus and anterior pituitary of male Siberian hamsters would be of interest. In addition, as this species is very sensitive to changes in environmental light, these studies were carried out in animals kept under short or long daily photoperiods. The results of this investigation show that in male Siberian hamsters, photoperiod did not markedly influence the hypothalamic concentrations of substance P- and neurokinin A-like immunoreactive substances, which steadily increased from prepuberty up to 40-50 days of age. The influence of photoperiod, however, was much more marked on tachykinin levels in the anterior pituitary gland, because the developmental increase of tachykinins in this gland was significantly more evident in animals kept under short daily photoperiods than in animals under long daily exposure to light. These results are similar to those previously obtained in female Siberian hamsters, in spite of the fact that estradiol and testosterone seem to have opposite effects on anterior pituitary tachykinin concentrations. It seems, therefore, that, in addition to gonadal steroids, other factors may be important mediators of the tachykinin response to photoperiods in the anterior pituitary of the male Siberian hamster.
Aging↗
Influence of hypothalamus and ovary on pituitary function in transgenic mice expressing the bovine growth hormone gene and in growth hormone-deficient Ames dwarf mice.
Female transgenic mice expressing the bovine (b) growth hormone (GH) gene exhibit reduced fertility, and GH-deficient dwarf mice are sterile. However, in these animals, the influence of the hypothalamus and ovary on pituitary functions is poorly understood. To address this issue, female Ames dwarf mice received twice-daily injections of either vehicle or bGH (25 micrograms/injection per mouse) for 12 days. Vehicle-injected normal female siblings served as controls. On Day 7, these mice and adult female transgenic mice expressing the bGH gene with mouse metallothionein-I promoter, as well as their normal littermates, were ovariectomized. Two experiments were conducted with each group of mice: on Day 8 after ovariectomy of transgenic mice and their normal siblings, and on Day 4 after ovariectomy of dwarf mice and their normal siblings. In experiment I, mice were primed with 0.5 micrograms estradiol benzoate (EB) in oil and 24 h later received injections of 5 micrograms EB/100 g BW. The next day, these mice received injections of either saline or GnRH (1 ng/g BW), and 15 min later blood was obtained via heart puncture under anesthesia. In experiment II, ovariectomized mice either received injections of oil or were primed with 0.5 micrograms EB and 24 h later treated with 10 micrograms EB/100 g BW in oil. On the next day, blood was obtained as in experiment I. In transgenic mice and in their normal littermates, plasma bGH, FSH, LH, and prolactin levels were measured. In dwarf mice and in their normal siblings, plasma LH and insulin-like growth factor (IGF)-I levels were determined. Bovine GH was secreted in transgenic mice, but this hormone was not detectable in normal siblings. Expression of the GH gene resulted in attenuated (p < 0.001) FSH response to GnRH treatment, while LH response was similar to that in controls. The ovariectomy-induced increases in plasma FSH (p < 0.001) and LH (p < 0.05) levels were decreased in transgenic mice. The absolute circulating FSH levels were also decreased (p < 0.001) in estrogen-treated transgenic mice, while plasma LH level were similar in normal mice and in mice bearing the bGH gene. The ovariectomized transgenic mice were hyperprolactinemic. In ovariectomized dwarf mice, IGF-I was not detectable, and GH treatment induced its secretion. In these mice, plasma LH concentrations were reduced (p < 0.05), and GH treatment increased (p < 0.01) LH secretion. In oil-injected ovariectomized dwarf mice, the plasma LH response to GnRH treatment was reduced (p < 0.001), and GH administration normalized this response. The negative feedback effect of estrogen on LH secretion was decreased (p < 0.025) in dwarf mice. These indicate that the alterations in gonadotropin secretion are dependent on the duration of exposure of neuroendocrine system to bGH and that bGH alters the effects of GnRH and estrogen on gonadotropin secretion in these two widely divergent experimental mouse models.
Animals↗
Testicular gamma-aminobutyric acid and circulating androgens in Syrian and Djungarian hamsters during sexual development.
Several factors, besides luteinizing hormone (LH), participate in the modulation of testicular function. A number of neurotransmitters are reported to be involved in this process, including a stimulatory action of gamma-aminobutyric acid (GABA) on steroidogenesis in the rat testis. The purpose of this study was to investigate the testicular pattern of GABA and glutamic acid, one of its main precursors, during sexual maturation in two seasonally breeding species: Syrian (golden) and Djungarian hamsters. Plasma androgen levels were also measured. The animals were maintained under long-day photoperiod (16:8, L:D) and were killed at 23, 30, 36, 46, 60, and 90 days of age. A different pattern of developmental changes in body and testicular weight was observed in these two species. GABA was present in the testes at all ages studied. GABA concentration and content showed a sharp elevation in the prepubertal period in golden as well as Djungarian hamsters. However, glutamic acid concentrations remained nearly constant during development in both species. Glutamic acid content increased gradually with age in the golden hamster, while a marked peak at 36 days of age was detected in the Djungarian hamster. Plasma testosterone and dihydrotestosterone levels were maximal at pubertal age in both species. The plasma levels of 5 alpha-androstane-3 alpha, 17 beta-diol increased significantly at 30 days of age in the golden hamster while in Djungarian hamsters this steroid remained unchanged. These results suggest that glutamic acid may serve as a precursor for GABA biosynthesis in the testis. In addition, changes in testicular GABA and plasma androgen levels might reflect a modulatory effect of this neurotransmitter in the acquisition of steroidogenic capability during development.
Androgens↗
Systemically administered gonadotrophin-releasing hormone enhances copulatory behaviour in castrated, testosterone-treated hyperprolactinaemic male rats.
Hyperprolactinaemic male rats exhibit deficits in copulatory behaviour which can be reversed by a single injection of GnRH. We tested whether systemically administered GnRH can stimulate copulatory behaviour independently of LH-mediated increases in plasma testosterone levels. Gonadectomized, pituitary-grafted adult male Fischer 344 rats bearing implants of 5, 10 or 20 mm capsules of testosterone were administered a single injection of 500 ng GnRH or saline s.c., 30 min prior to copulation tests. Pituitary-grafted castrates displayed copulatory deficits, relative to sham-operated castrates with identical levels of testosterone replacement. Administration of 500 ng GnRH to pituitary-grafted castrates bearing 10 mm testosterone implants significantly increased the proportion of rats that mounted, intromitted and ejaculated during a 30 min test. This treatment also reduced significantly the latency of intromission and ejaculation, and increased significantly the frequency of intromission. The copulatory behaviour of the sexually unresponsive, pituitary-grafted castrates bearing 5 mm testosterone implants, or of the more sexually responsive castrates bearing 20 mm testosterone implants, was not altered significantly by GnRH injections. These results support the hypothesis that copulatory deficits in moderately hyperprolactinaemic rats are due in part to reduced hypothalamic GnRH release, and suggest that GnRH can stimulate sexual behaviour in these animals via mechanisms that are independent of luteinizing hormone-induced testosterone release. However, a threshold level of testosterone (achieved with 10 mm implants) appears to be required for GnRH to elicit this effect.
Animals↗
Developmental aspects of prolactin receptor gene expression in fetal and neonatal mice.
The prolactin receptor (PRL-R), a member of the hematopoietin cytokine receptor superfamily, is widely distributed among mammalian tissues. To understand better the potential sites of action and onset of potential PRL responsiveness, the developmental distribution pattern of PRL-R mRNA expression in fetal and neonatal mice was examined. Fetal mouse tissues were collected at distinct stages from timed pregnancies. Following extraction of total RNA, onset of gene expression was evaluated via reverse transcription-polymerase chain reaction (RT-PCR) and Southern hybridization was employed for verification. Expression of PRL-R mRNA was first observed on day 14 in the liver and cranium and on day 15 in the kidney, lung and thymus gland. Pituitary and adrenal glands were positive for PRL-R at day 18 of gestation through to day 1 of postnatal life. Neither whole fetuses prior to day 14 (days 10-13) of gestation nor skin and bladder tissues from 2-day-old mice generated detectable RT-PCR signals for PRL-R. The presence of PRL-R mRNA in fetal thymus and spleen tissues suggests a possible role for PRL in the development of the immune system. Prolactin may act directly on the pituitary to influence its own secretion and/or that of other pituitary-derived factors, as evidenced by the presence of PRL-R mRNA in the pituitary glands of fetal and 1-day-old mice. These data are the first to show the presence of PRL-R gene expression in various organ systems in fetal mice and suggest that PRL is among several factors necessary to coordinate developmental activities.
Animals↗
Age-related effects of ectopic pituitary transplants on the activation of Ames dwarf mouse lymphocytes in vitro.
Prolactin (PRL), one of the anterior pituitary hormones, has been implicated in the development and maintenance of immune system function. The following experiments were conducted to evaluate age-related effects of PRL on immune system activity in a PRL-deficient mouse model, the Ames dwarf. Two- and ten-month-old dwarf and phenotypically normal male mice were used. Six dwarf mice from each age group received a surgically implanted pituitary (under the kidney capsule) from a normal donor female mouse. Six different dwarfs were similarly operated but had no pituitary graft inserted and will be referred to as sham-operated dwarfs. Three weeks following surgery, spleens were removed from the dwarfs and age-matched normal mice and splenocytes isolated. The two age groups will subsequently be referred to as "3-month-old" and "11-month-old", respectively, The splenocytes were used in mitogen-induced (concanavalin A (Con A); phytohemagglutinin (PHA); lipopolysaccharide (LPS)) proliferation assays, and histopaque isolated lymphocytes were used for T cell surface marker determination. Pituitary grafting increased body weights of dwarf mice although the weights were less than control mice independent of age. A similar pattern was observed for total number of splenocytes per spleen. In young animals, the relative number of splenocytes per gram of body weight increased in pituitary grafted dwarfs reaching values of control mice, whereas much smaller differences were observed in older animals. The relative percentage of CD4+ cells was reduced (P<0.01) in 3-month-old pituitary-grafted mice, compared with sham-operated dwarf mice, while no differences were observed between sham- operated dwarf and normal mice. Pituitary grafting did not affect the numbers of CD8+ cells. In 11-month-old animals, the relative percentages of CD4+ and CD8+ cells were greater (P<0.05) in sham-operated dwarf than in normal mice but not affected by pituitary grafting. At 3 months of age, proliferation of splenocytes in response to Con A was increased (P<0.05) in sham-operated dwarfs and reduced (P<0.05) in pituitary-grafted dwarfs compared with normal mice. In contrast, PHA stimulation of splenocytes was decreased (P<0.05) in sham-operated compared with normal, and was still lower in mice receiving an ectopic pituitary. Substantially different responses were obtained in 11-month-old animals. In response to Con A, splenocytes from sham-operated dwarfs exhibited a reduced (P<0.05) proliferative capacity as compared with normals, and proliferation was increased in pituitary-grafted as compared with sham-operated dwarfs. In the presence of PHA, the proliferative capacity of splenocytes was greater (P<0.05) in sham-operated dwarfs as compared to normals and pituitary grafting normalized this parameter. These results demonstrate differential effects of PRL deficiency (sham-operated dwarfs versus normal) and of PRL replacement (pituitary-grafted versus sham-operated) in young as compared with old dwarf mice on immune system activation.
Age Factors↗
Effects of growth hormone on neuroendocrine function.
Although the role of growth hormone (GH) in the control of reproductive functions is not well understood, there is considerable evidence that the states of both GH deficiency and GH excess are typically associated with reproductive deficits. To identify the possible involvement of functional alterations in the hypothalamic-pituitary system in producing these deficits, we are studying neuroendocrine function related to reproduction in transgenic animals overexpressing GH, in animals with congenital GH deficiency, and in animals with selective immunoneutralization of GH. The results indicate that GH acts on the hypothalamus to alter dopaminergic and noradrenergic control of prolactin and gonadotropin release. Life-long elevation of GH levels outside the physiological ranges disrupts feedback control of luteinizing hormone (LH) release by gonadal streroids. Plasma LH and follicle-stimulating hormone (FSH) levels and feedback control of LH release are also abnormal in GH-deficient animals indicating that physiological levels of endogenous GH are normally involved in the control of gonadotropin release. Differences between the effects of bovine vs. human GH in transgenic mice and differential effects of GH deficiency in mice and rats should facilitate identification of the mechanisms involved in the actions of GH on the hypothalamic-pituitary system.
Animals↗
Oxytocin and prolactin release after hypertonic saline administration in melatonin-treated male Syrian hamsters.
The aim of the present investigations was to examine the effects of melatonin (Mel) on oxytocin (OT) release under conditions of osmotic stimulation, brought about by hypertonic saline administration, as well as to determine whether osmotically stimulated OT release in Mel-treated Syrian hamster is associated with alterations in the release of prolactin (PRL) and in norepinephrine (NE) and dopamine (DA) content in the hypothalamus. In both Mel- and vehicle-treated hamsters, injection of hypertonic saline was followed by a significant decrease in OT content in the pituitary neurointermediate lobe (NIL) and elevation of plasma OT and PRL levels. Melatonin injections had no significant affect on NIL OT content in either isotonic- or hypertonic-saline treated animals. Pretreatment with Mel did not alter plasma OT or PRL levels in isotonic saline-injected animals. However, Mel facilitated the release of OT, but prevented the release of PRL after hypertonic saline administration. Melatonin treatment reduced hypothalamic NE content (but not that of DA) in isotonic-saline treated animals. After osmotic stimulation, hypothalamic content of NE and DA was significantly lower in Mel-treated than in vehicle-treated animals. Data from the present study suggest that the osmotically-stimulated release of OT and PRL seems to be related to the activation of noradrenergic rather than dopaminergic transmission. Both dopaminergic and noradrenergic transmission may be, however, involved in mediating the effects of Mel on the osmotically-activated OT and PRL release.
Analysis of Variance↗
Genetic determination of coat color affects testicular steroidogenesis in the Mustela vison.
Coat color genes in mammals are known to be developmental genes with wide pleiotropic effects. The present study was undertaken to study testicular steroidogenesis in American Mink (Mustela vison) of various coat color phenotypes. No differences in testicular steroid levels were observed between fertile and infertile mink with the standard phenotype and genotype (BB jj MM PP). Mink with the opaline phenotype and genotype (bb mm pp), were found to have in their testes, 20-40% higher levels of progesterone, five times higher levels of 17-hydroxyprogesterone, and eight times higher levels of testosterone, than the corresponding values in other mink. No other differences were observed among the different types of mink. Since the genotype of the opaline mink differs from the other mink studied, only in their combination at the pastel (b) and moyle (m) loci, their bb mm genotype could be assumed to be responsible for the increase in testicular steroids.
17-alpha-Hydroxyprogesterone↗
Melatonin inhibits oxytocin and vasopressin release from the neurointermediate lobe of the hamster pituitary.
The aim of this investigation was to study whether melatonin affects the release of oxytocin and vasopressin by the pituitary neurointermediate lobe of the Syrian hamster in vitro. The effect of melatonin was studied on the unstimulated (pre- and post-K+ -stimulated) release of oxytocin and vasopressin and on the response to K+ stimulation. Melatonin significantly inhibited unstimulated release of these hormones in all concentrations (10(-11) M, 10(-9) M and 10(-7) M) studied. K+ -stimulated release of oxytocin and vasopressin was significantly decreased by the 10(-9) M dose of melatonin. It is concluded that melatonin is active in modifying the release of these peptides in the Syrian hamster neurointermediate lobe, as it has been previously demonstrated in the rat hypothalamus.
Animals↗
In vivo and in vitro effects of neuropeptide K and neuropeptide gamma on the release of growth hormone.
There is anatomical and experimental evidence suggesting that tachykinins have a role in the regulation of secretion of anterior pituitary hormones. In this investigation, the effects of neuropeptide K and neuropeptide gamma on the secretion of GH were studied in vivo and in vitro. Injections of neuropeptide K into the third ventricle of freely moving, ovariectomized rats resulted in a significant increase of plasma GH, but neuropeptide gamma induced no significant changes in these levels, although it did induce a significant increase in GH pulse height. In vitro, neither neuropeptide K nor neuropeptide gamma had any apparent effect on GH release from hemipituitaries incubated for 2 h. These results suggest that neuropeptide K may have a modulatory function in the regulation of GH secretion from the anterior pituitary, through an action exerted at the hypothalamic level, and the effects of neuropeptide gamma seem to be more marginal.
Animals↗
The Ames dwarf gene is required for Pit-1 gene activation.
The tissue-specific POU domain transcription factor Pit-1 is required for phenotypic specification of three cell types in the anterior pituitary gland of mammals: somatotropes, thyrotropes, and lactotropes. Mutations in the Pit-1 gene, as first described in Snell and Jackson dwarf (dw) mice, led to pituitary hypoplasia due to lack of these three cell types, as well as hypothyroidism and dwarfism because of deficiency of thyroid-stimulating hormone and growth hormone, respectively. The Ames dwarf (df) exhibits a phenotype identical to that of Pit-1 mutated mice but the defective gene has been mapped to a locus on mouse chromosome 11, distinct from the Pit-1 gene located on mouse chromosome 6. Our studies indicate that initial activation of the Pit-1 gene is deficient in the Ames dwarf. This suggests that the df gene is required for activation of the Pit-1 gene and provides evidence for a hierarchy of tissue-specific factors required for cellular commitment in the anterior pituitary gland.
Animals↗
Neuroendocrine control of reproduction.
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Animals↗
Effects of photoperiod on testicular inhibin-alpha and androgen binding protein mRNA expression during postnatal development in Siberian hamsters, Phodopus sungorus.
The effects of short photoperiod (SPP) on testicular inhibin-alpha and androgen binding protein (ABP) mRNA expression were investigated in Siberian hamsters during postnatal development. Hamsters were raised in either long photoperiod (LPP; 16L:8D) or in a SPP (6L:18D). Hamsters were sacrificed at the following developmental ages: preweaning (18-20 days), immature (28-30 days), pubertal (38-40 days), postpubertal (48-50 days) and adult (60-62 days; n = 7-12 animals per group). Body and testicular weights were determined and plasma FSH levels were measured by validated RIA. As expected, testicular weight and circulating FSH levels were significantly lower in hamsters raised in SPP than in LPP animals. Photoperiod had no significant effect on body weight except during postpubertal age. The northern analyses of inhibin-alpha and ABP expression in the testes revealed that the corresponding relative mRNA levels were higher in animals exposed to SPP than in animals from LPP. These findings suggest that the testes of hamsters exposed to a SPP can synthesize inhibin-alpha and ABP, and we speculate that the release of these proteins may be regulated by photoperiod and the role of FSH in the control of inhibin-alpha and ABP gene expression in male Siberian hamsters may be minimal.
Androgen-Binding Protein↗