No abstract

DNA methylation is an important epigenetic modification regulating various biological phenomena, including genomic imprinting and transposon silencing. It is known that methylation of the differentially methylated regions (DMRs) associated with paternally imprinted genes and of some repetitive elements occurs during male germ cell development in the mouse. We have performed a detailed methylation analysis of the paternally methylated DMRs (H19, Dlk1/Gtl2 and Rasgrf1), interspersed repeats [SineB1, intracisternal A particle (IAP) and Line1] and satellite repeats (major and minor) to determine the timing of this de novo methylation in male germ cells. Furthermore, we have examined the roles of the de novo methyltransferases (Dnmt3a and Dnmt3b) and related protein (Dnmt3L) in this process. We found that methylation of all DMRs and repeats occurred progressively in fetal prospermatogonia and was completed by the newborn stage. Analysis of newborn prospermatogonia from germline-specific Dnmt3a and Dnmt3b knockout mice revealed that Dnmt3a mainly methylates the H19 and Dlk1/Gtl2 DMRs and a short interspersed repeat SineB1. Both Dnmt3a and Dnmt3b were involved in the methylation of Rasgrf1 DMR and long interspersed repeats IAP and Line1. Only Dnmt3b was required for the methylation of the satellite repeats. These results indicate both common and differential target specificities of Dnmt3a and Dnmt3b in vivo. Finally, all these sequences showed moderate to severe hypomethylation in Dnmt3L-deficient prospermatogonia, indicating the critical function and broad specificity of this factor in de novo methylation.

Host-defense mechanisms against transposable elements are critical to protect the genome information. Here we show that tudor-domain containing 9 (Tdrd9) is essential for silencing Line-1 retrotransposon in the mouse male germline. Tdrd9 encodes an ATPase/DExH-type helicase, and its mutation causes male sterility showing meiotic failure. In Tdrd9 mutants, Line-1 was highly activated and piwi-interacting small RNAs (piRNAs) corresponding to Line-1 were increased, suggesting that feedforward amplification operates in the mutant. In fetal testes, Tdrd9 mutation causes Line-1 desilencing and an aberrant piRNA profile in prospermatogonia, followed by cognate DNA demethylation. TDRD9 complexes with MIWI2 with distinct compartmentalization in processing bodies, and this TDRD9-MIWI2 localization is regulated by MILI and TDRD1 residing at intermitochondrial cement. Our results identify TDRD9 as a functional partner of MIWI2 and indicate that the tudor-piwi association is a conserved feature, while two separate axes, TDRD9-MIWI2 and TDRD1-MILI, cooperate nonredundantly in the piwi-small RNA pathway in the mouse male germline.

Objective: The development of computed tomography (CT) and magnetic resonance imaging (MRI) has resulted in the discovery of unsuspected endocrinologically silent pituitary masses (pituitary incidentalomas). The aim of this study was to perform a national survey on pituitary incidentalomas in order to establish an appropriate approach to them. Design and methods: Five hundred and six patients with pituitary incidentalomas were obtained by questionnaire from March 1999 to May 2000 under the auspices of the Ministry of Health, Labor and Welfare in Japan. Two hundred and fifty-eight patients underwent surgery (surgical group), while 248 patients were followed up conservatively for a mean period of 26.9 months (range 6-173 months) (non-surgical group). Clinical and biochemical assessment, CT or MRI of the pituitary, and visual field testing by Goldman perimetry were assessed at baseline and 6 months and yearly thereafter. Results: Thirty-three patients with pituitary incidentalomas (13.3%) developed tumor enlargement during the mean follow-up period of 45.5 months. Of 115 estimated non-functioning adenomas, 23 tumors (20.0%) increased during a mean follow-up period of 50.7 months (range 10 -173 months), while 5 of 94 (5.3%) estimated Rathke's cysts increased in size during follow-up. Pituitary apoplexy occurred in one of 248 patients (0.4%). Conclusions: Pituitary incidentalomas usually follow a benign course. We recommend transsphenoidal adenectomy for a solid mass attached to the optic chiasma estimated to be a pituitary adenoma by MRI. Other patients should be followed up by MRI every 6 months for the first 2 years, and then yearly.

Abstract:Recently, erythropoietin (EPO) receptors and synthesis of EPO have been identified in the brain. To clarify the effects of EPO on neuronal cells, we investigated the effects of EPO on Ca 2ϩ uptake, intracellular Ca 2ϩ concentration, membrane potential, cell survival, release and biosynthesis of dopamine, and nitric oxide (NO) production in differentiated PC12 cells, which possess EPO receptors. EPO (10 Ϫ12 -10 Ϫ10 M) increased 45 Ca 2ϩ uptake and intracellular Ca 2ϩ concentration in PC12 cells in a dose-related manner; these increases were inhibited by nicardipine (1 M) or anti-EPO antibody (1:100 dilution). EPO induced membrane depolarization in PC12 cells. After a 5-day culture without serum and nerve growth factor (NGF), viable cell number decreased to 50% of that of the control cells cultured with serum and NGF. EPO (10 Ϫ13 -10 Ϫ10 M) increased the number of viable cells cultured without serum and NGF; this increase was blunted by nicardipine or anti-EPO antibody. Incubation with EPO (10 Ϫ13 -10 Ϫ10 M) stimulated mitogen-activated protein kinase activity in PC12 cells. EPO (10 Ϫ13 -10 Ϫ10 M) increased dopamine release from PC12 cells and tyrosine hydroxylase activity; these increases were sensitive to nicardipine or anti-EPO antibody. Following a 4-h incubation with EPO (10 Ϫ14 -10 Ϫ10 M), NO production was increased, which was blunted by nicardipine and anti-EPO antibody. In contrast, maximal NO synthase activity was not changed by EPO. These results suggest that EPO stimulates neuronal function and viability via activation of Ca 2ϩ channels. Key Words: Erythropoietin-PC12 cells-Ca 2ϩ channels-Cell survivalDopamine release -Nitric oxide.

Synthetic vasoactive intestinal polypeptide (VIP) administered either intraventricularly or iv caused a significant and dose-related increase in plasma PRL levels in urethane-anesthetized rats. The administration of naloxone, an opiate receptor antagonist, significantly blunted the plasma PRL response to VIP. Increases in plasma PRL induced by VIP were also significantly suppressed by L-dopa, a precursor of dopamine, whereas pilocarpine, a cholinergic agonist, diphenhydramine, a histamine antagonist, and cyproheptadine, an antiserotoninergic agent, did not affect the plasma PRL response to VIP. In in vitro experiments, VIP alone did not stimulate PRL release from cultured pituitary cells, but it significantly attenuated the inhibitory action of dopamine, which was not blocked by naloxone. These results suggest that VIP stimulates rat PRL secretion, at least in part, through activation of an opiate receptor in the central nervous system and by blocking the inhibitory action of a dopaminergic mechanism at the pituitary level.

The effects of thyrotropin-releasing hormone (TRH) on the release of growth hormone (GH), prolactin (PRL) and thyrotropin (TSH) were investigated in patients with depression. Intravenous injection of synthetic TRH (500 mug) caused a significant increase in plasma GH (peak value: 7.7 minus 35.0 ng/ml) in 8 of 13 patients with mental depression. After clinical recovery these patients had no response of plasma GH to TRH. TRH administration did not raise plasma GH in normal subjects examined. Plasma PRL responses to TRH were significantly enchanced (P smaller than 0.05) in depressed patients compared with control subjects. Plasma TSH responses to TRH were significantly blunted in patients with depression (P smaller than 0.05). These results suggest disorders in the hypothalamo-pituitary function in depression.

NANOS2 is an RNA-binding protein essential for fetal male germ cell development. While we have shown that the function of NANOS2 is vital for suppressing meiosis in embryonic XY germ cells, it is still unknown whether NANOS2 plays other roles in the sexual differentiation of male germ cells. In this study, we addressed the issue by generating Nanos2/Stra8 double knockout (dKO) mice, whereby meiosis was prohibited in the double-mutant male germ cells. We found that the expression of male-specific genes, which was decreased in the Nanos2 mutant, was hardly recovered in the dKO embryo, suggesting that NANOS2 plays a role in male gene expression other than suppression of meiosis. To investigate the molecular events that may be controlled by NANOS2, we conducted a series of microarray analyses to search putative targets of NANOS2 that fulfilled 2 criteria: (1) increased expression in the Nanos2 mutant and (2) the mRNA associated with NANOS2. Interestingly, the genes predominantly expressed in undifferentiated primordial germ cells (PGCs) were significantly selected, implying the involvement of NANOS2 in the termination of the characteristics of PGCs. Furthermore, we showed that NANOS2 is required for the maintenance of mitotic quiescence, but not for the initiation of the quiescence in fetal male germ cells. These results suggest that NANOS2 is not merely a suppressor of meiosis, but instead plays pivotal roles in the sexual differentiation of male germ cells.