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In mammalian cloning by somatic cell nuclear transfer (SCNT), the treatment of reconstructed embryos with histone deacetylase (HDAC) inhibitors improves efficiency. So far, most of those used for SCNT are hydroxamic acid derivatives-such as trichostatin A-characterized by their broad inhibitory spectrum. Here, we examined whether mouse SCNT efficiency could be improved using chlamydocin analogues, a family of newly designed agents that specifically inhibit class I and IIa HDACs. Development of SCNT-derived embryos in vitro and in vivo revealed that four out of five chlamydocin analogues tested could promote the development of cloned embryos. The highest pup rates (7.1-7.2%) were obtained with Ky-9, similar to those achieved with trichostatin A (7.2-7.3%). Thus, inhibition of class I and/or IIa HDACs in SCNT-derived embryos is enough for significant improvements in full-term development. In mouse SCNT, the exposure of reconstructed oocytes to HDAC inhibitors is limited to 8-10 h because longer inhibition with class I inhibitors causes a two-cell developmental block. Therefore, we used Ky-29, with higher selectivity for class IIa than class I HDACs for longer treatment of SCNT-derived embryos. As expected, 24-h treatment with Ky-29 up to the two-cell stage did not induce a developmental block, but the pup rate was not improved. This suggests that the one-cell stage is a critical period for improving SCNT cloning using HDAC inhibitors. Thus, chlamydocin analogues appear promising for understanding and improving the epigenetic status of mammalian SCNT-derived embryos through their specific inhibitory effects on HDACs., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society for the Study of Reproduction.)

Study Question: Is the poor development of embryos generated from round spermatid injection (ROSI) in humans and animals associated with abnormal active DNA demethylation?, Summary Answer: A significant proportion of ROSI-derived embryos failed to undergo active DNA demethylation., What Is Known Already: Active DNA demethylation is initiated by the conversion of 5-methylcytosine (5mC) to 5-hydroxycytosine (5hmC) by the Tet3 enzyme. Active demethylation proceeds in a more pronounced manner in the male pronucleus than in the female one., Participants/materials, Setting, Methods: Mouse zygotes generated by ICSI or ROSI were analyzed for active DNA methylation by quantification of 5mC and 5hmC using specific antibodies. Some ROSI-derived embryos were subjected to time-lapse imaging for DNA methylation levels and were transferred into recipient pseudo-pregnant female mice., Main Results and the Role of Chance: In ICSI-derived embryos, the male:female pronucleus (M/F) ratio of 5mC immunostaining intensity was decreased while that of 5hmC was increased. However, a significant proportion of ROSI-derived embryos showed unchanged M/F ratios for 5mC and 5hmC even at the late zygotic period, indicating that they failed to undergo asymmetric active DNA demethylation. Consistent with this, some ROSI-derived embryos did not show preferential localization of Tet3 to the male pronucleus. ROSI-derived embryos were classified into 'demethylated' or 'non-demethylated' groups by time-lapse imaging and transferred into recipient female mice separately. More normal-sized fetuses were retrieved from the 'demethylated' group than 'non-demethylated' group at Day 11.5 of pregnancy., Limitations, Reasons for Caution: A causal relationship between impaired active DNA demethylation and the poor developmental ability of ROSI-derived embryos remains to be determined., Wider Implications of the Findings: We identified two types of ROSI-derived embryos in terms of the degree of active DNA demethylation. Induction of normal DNA demethylation at the zygotic stage might help in the technical improvement of ROSI., Study Funding/competing Interests: The work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by the RIKEN Epigenetics Program. The authors have no competing interests to declare., (© The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Whereas cloning mammals by direct somatic cell nuclear transfer has been successful using a wide range of donor cell types, neurons from adult brain remain "unclonable" for unknown reasons. Here, using a combination of two epigenetic approaches, we examined whether neurons from adult mice could be cloned. First, we used a specific antibody to discover cell types with reduced amounts of a repressive histone mark-dimethylated histone H3 lysine 9 (H3K9me2)-and identified CA1 pyramidal cells in the hippocampus and Purkinje cells in the cerebellum as candidates. Second, reconstructed embryos were treated with trichostatin A (TSA), a potent histone deacetylase inhibitor. Using CA1 cells, cloned offspring were obtained at high rates, reaching 10.2% and 4.6% (of embryos transferred) for male and female donors, respectively. Cerebellar Purkinje cell nuclei were too large to maintain their genetic integrity during nuclear transfer, leading to developmental arrest of embryos. However, gene expression analysis using cloned blastocysts corroborated a high rate of genomic reprogrammability of CA1 pyramidal and Purkinje cells. Neurons from the hippocampal dentate gyrus and cerebral cortex, which had higher amounts of H3K9me2, could also be used for producing cloned offspring, but the efficiencies were low. A more thorough analysis revealed that TSA treatment was essential for cloning adult neuronal cells. This study demonstrates, to our knowledge for the first time, that adult neurons can be cloned by nuclear transfer. Furthermore, our data imply that reduced amounts of H3K9me2 and increased histone acetylation appear to act synergistically to improve the development of cloned embryos., (© 2015 by the Society for the Study of Reproduction, Inc.)

In mice, the establishment of paternal genomic imprinting in male germ cells starts at midgestation, as suggested by DNA methylation analyses of differentially methylated regions (DMRs). However, this information is based on averages from mixed populations of germ cells, and the DNA methylation pattern might not always provide a full representation of imprinting status. To obtain more detailed information on the establishment of paternal imprinting, single prospermatogonia at Embryonic Days 15.5 (E15.5), E16.5, and E17.5 and at Day 0.5 after birth were cloned using nuclear transfer; previous reports suggested that cloned embryos reflected the donor's genomic imprinting status. Then, the resultant fetuses (E9.5) were analyzed for the DNA methylation pattern of three paternal DMRs (IG-DMR, H19 DMR, and Rasgrf1 DMR) and the expression pattern of imprinted genes therein. The overall data indicated that establishment of genomic imprinting in all paternally imprinted regions was completed by E17.5, following a short intermediate period at E16.5. Furthermore, comparison between the methylation status of DMRs and the expression profiles of imprinted genes suggested that methylation of the IG-DMR, but not the H19 DMR, solely governed the control of its imprinted gene cluster. The Rasgrf1 DMR seemed to be imprinted later than the other two genes. We also found that the methylation status of the Gtl2 DMR, the secondary DMR that acquires DNA methylation after fertilization, was likely to follow the methylation status of the upstream IG-DMR. Thus, the systematic analyses of prospermatogonium-derived embryos provided additional important information on the establishment of paternal imprinting., (© 2014 by the Society for the Study of Reproduction, Inc.)

Somatic cell nuclear transfer (SCNT) is a unique technology that produces cloned animals from single cells. It is desirable from a practical viewpoint that donor cells can be collected noninvasively and used readily for nuclear transfer. The present study was undertaken to determine whether peripheral blood cells freshly collected from living mice could be used for SCNT. We collected a drop of peripheral blood (15-45 μl) from the tail of a donor. A nucleated cell (leukocyte) suspension was prepared by lysing the red blood cells. Following SCNT using randomly selected leukocyte nuclei, cloned offspring were born at a 2.8% birth rate. Fluorescence-activated cell sorting revealed that granulocytes/monocytes and lymphocytes could be roughly distinguished by their sizes, the former being significantly larger. We then cloned putative granulocytes/monocytes and lymphocytes separately and obtained 2.1% and 1.7% birth rates, respectively (P > 0.05). Because the use of lymphocyte nuclei inevitably results in the birth of offspring with DNA rearrangements, we applied granulocyte/monocyte cloning to two genetically modified strains and two recombinant inbred strains. Normal-looking offspring were obtained from all four strains tested. The present study clearly indicated that genetic copies of mice could be produced using a drop of peripheral blood from living donors. This strategy will be applied to the rescue of infertile founder animals or a "last-of-line" animal possessing invaluable genetic resources.

In the present study, we demonstrated that the mouse embryonic stem cells were differentiated into ciliated epithelial cells, with characteristics of normal ciliated cells. These cells expressed ciliary marker proteins, such as beta-tubulin IV and hepatocyte nuclear factor-3/forkhead homolog 4 (HFH-4), and processed microtubules were arranged in the 9 + 2 structure, which is the same specific alignment observed in normal ciliary microtubules. The cilia of these cells were beating at a frequency of 17-20 Hz. The differentiated embryoid bodies (EBs) containing these ciliated cells expressed respiratory marker genes such as thyroid transcription factor-1 and surfactant protein-C. For the induction of ciliated cells, culture of EBs in serum-free medium during the initial 2 days of the attachment was indispensable. When EBs were treated with bone morphogenetic proteins, the expression of HFH-4 was decreased, and the ciliated cells were scarcely differentiated. Previous methods for inducing ciliated cells in vitro from embryonic or adult tissues involved an air-liquid interface. The system used in this study more closely mimics the normal development of ciliated cells; thus, an added advantage of the system is as a tool for studying the differentiation mechanism of normal ciliated epithelial cells.

The hypercalcemia of various granulomatoses is caused by endogenous 1,25-dihydroxyvitamin D [1,25-(OH)2D3] overproduction by disease-activated macrophages. The inability of 1,25(OH)2D3 to suppress its synthesis in macrophages contrasts with the tight control of its production in macrophage precursors, peripheral blood monocytes (PBM). We examined whether 1,25(OH)2D3 resistance develops as PBM differentiate to macrophages or with macrophage activation. Normal human pulmonary alveolar macrophages (PAM) are less sensitive to 1,25(OH)2D3 than PBM, despite similar vitamin D receptor content; however, both PBM and PAM respond to exogenous 1,25-(OH)2D3 by inhibiting 1,25(OH)2D3 synthesis and inducing 1,25(OH)2D3 degradation through enhancement of 24-hydroxylase mRNA levels and activity. The human monocytic cell line THP-1 mimics PAM in 1,25(OH)2D3 synthesis and sensitivity to exogenous 1,25(OH)2D3. We utilized THP-1 cells to examine the response to 1,25(OH)2D3 with macrophage activation. Activation of THP-1 cells with gamma-interferon (gamma-IFN) enhances 1,25(OH)2D3 synthesis 30-fold, blocks 1,25-(OH)2D3 suppression of its synthesis, and reduces by 42.2% 1,25-(OH)2D3 induction of its degradation. The antagonistic effects of gamma-IFN are not merely restricted to enzymatic activities. In THP-1 cells and in normal PBM, gamma-IFN inhibits 1,25-(OH)2D3 induction of 24-hydroxylase mRNA levels without reducing mRNA stability, suggesting gamma-IFN inhibition of 1,25(OH)2D3 transactivating function. These results explain 1,25(OH)2D3 overproduction in granulomatoses and demonstrate potent inhibition by gamma-IFN of 1,25(OH)2D3 action in immune cells.

22-Oxacalcitriol [1,25-(OH)2-22oxa-D3] mimics the action of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] in a variety of target tissues, including the systemic control of calcitriol metabolism. Similar to 1,25-(OH)2D3, 1,25-(OH)2-22oxa-D3 decreases the rate of 1,25-(OH)2D3 synthesis and accelerates its metabolic clearance rate. We have previously shown that in normal human monocytes, physiological concentrations of 1,25-(OH)2D3 and 1,25-(OH)2-22oxa-D3 determine identical suppression of 1,25-(OH)2D3 synthesis. Moreover, both sterols have a similar potency to induce vitamin D degradation through stimulation of the C24-hydroxylation pathway. In this study, we examined the ability of normal human monocytes to metabolize 1,25-(OH)2-22oxa-D3 and whether the enzymes involved are the same as those that catabolize 1,25-(OH)2D3. Time-course experiments demonstrated no detectable basal catabolic activity. However, exogenous 1,25-(OH)2D3 at physiological concentrations induced 1,25-(OH)2-22oxa-D3 degradation by normal human monocytes. Competition experiments showed that a 10-fold molar excess of unlabeled 1,25-(OH)2D3 inhibited tritiated-1,25-(OH)2-22oxa-D3 catabolism by 85%, whereas a 10-fold excess of unlabeled 1,25-(OH)2-22oxa-D3 reduced tritiated-1,25-(OH)2-22oxa-D3 catabolism by 33%. In contrast, although a 10-fold excess of unlabeled 1,25-(OH)2D3 reduced tritiated 1,25-(OH)2D3 catabolism by 60%, a 1000-fold excess of 1,25-(OH)2-22oxa-D3 was required to reduce tritiated 1,25-(OH)2D3 catabolism to this degree. The apparent Km for 1,25-(OH)2-22oxa-D3 was significantly higher than that of 1,25-(OH)2D3 (2.0 +/- 0.8 0.9 +/- 0.2 nM, respectively; P < 0.001) for the catabolic pathway induced by physiological concentrations of 1,25-(OH)2D3. Moreover, the presence of 0.65 nM 1,25-(OH)2D3 caused an additional increase in the Km for 1,25-(OH)2-22oxa-D3 (3.2 +/- 0.8 nM). These data suggest that 1,25-(OH)2-22oxa-D3 may be less accessible than 1,25-(OH)2D3 to the hydroxylases involved in vitamin D catabolism. The resulting prolonged biological half-life of the analog in certain target tissues may be involved in its selectivity.

The microsomal ethanol-oxidizing system (MEOS) is a P450-dependent pathway for ethanol oxidation in hepatic microsomes. The bulk of MEOS activity is catalyzed by P450 2E1 (an ethanol-inducible isozyme of P450) in animal livers treated with ethanol. Rat brains also metabolize certain drugs, and it is theorized that a mechanism for drug metabolism exists within the brain which acts on P450. We compared immunohistochemical localization of P450 2E1 between ethanol-treated rats and pair-fed control rats. In control rats, immunoreactive P450 2E1 was detected in minute amounts in both basal ganglia and cerebellar cortices. After ethanol treatment, the content of P450 2E1 increased in the basal ganglia, and the enzyme was also induced in the cerebellar cortices, substantia nigra and hippocampus. We speculate that the rat brain metabolizes ethanol by P450 2E1.

1,25-Dihydroxyvitamin D3 (1,25D) regulates its own levels in circulation by affecting its rates of synthesis and degradation, 22-Oxacalcitriol (OCT), a vitamin D analog with low calcemic activity, decreases circulating PTH levels, one of the regulators of renal 1 alpha-hydroxylase, and stimulates vitamin D degradation in vitro. The purpose of this study was to examine the effects of OCT administration on serum levels of 1,25D. In normal rats, OCT administration (4-200 ng, ip, daily for 5 days) caused a dose-dependent reduction in serum calcitriol levels. At a dose of 200 ng, OCT reduced serum 1,25D from 34.5 +/- 2.7 to 10.9 +/- 0.7 pg/ml (P less than or equal to 0.01) without significant changes in ionized Ca or phosphorus levels. The contribution of the suppression of PTH by OCT to the reduction of serum 1,25D was examined by administering OCT to parathyroidectomized (PTX) rats. Two hundred nanograms of OCT, ip, daily for 5 days significantly reduced serum calcitriol from 29.7 +/- 7.6 to 9.1 +/- 0.5 pg/ml (P less than or equal to 0.01) in rats fed a normal calcium diet. Because OCT increased total calcium (TCa) in this group from 7.4 +/- 0.1 to 9.5 +/- 0.3 mg/dl, similar doses of OCT were given to PTX rats fed a calcium-deficient diet. OCT decreased 1,25D from 58.9 +/- 8.9 to 10.3 +/- 0.4 pg/ml and increased TCa from 4.8 +/- 0.2 to 7.4 +/- 0.1 mg/dl. Comparison of serum 1,25D for identical TCa levels in PTX rats (normal calcium diet controls vs. calcium-deficient diet, OCT-treated) clearly indicates that OCT per se reduced serum 1,25D. Further support for a direct effect of OCT was provided by studies in PTX rats fed a low phosphorus diet. OCT decreased serum 1,25D from 125.8 +/- 15.6 to 10.9 +/- 0.6 pg/ml without significant changes in TCa. To further characterize the mechanisms involved in this effect, similar studies were performed in six normal dogs. Intravenous administration of 0.75 micrograms OCT every other day for 1 week decreased serum calcitriol from 25.4 +/- 3.2 to 12.2 +/- 1.3 pg/ml (P less than or equal to 0.002). Ionized Ca and phosphorus remained unchanged. Despite the short half-life of OCT in the circulation, 1,25D levels returned to basal concentrations 96 h after the last dose of OCT.(ABSTRACT TRUNCATED AT 400 WORDS)

Using sea urchin (Hemicentrotus pulcherimus) sperm flagella, ATP hydrolysis coupled to sliding movement of microtubules was investigated. Flagellar axonemes were pretreated with trypsin and the microtubules induced to slide by addition of ATP (50-1,000 microM) at 0-20 degrees C. Motion-dependent hydrolysis of ATP was observed immediately after the addition of ATP, the rate of which was higher than that of steady state hydrolysis in axonemes without trypsin-treatment, or after complete disintegration. The rate of hydrolysis of ATP divided by the sliding velocity of microtubules reflects the ATP consumption necessary per unit distance of microtubule sliding. This parameter varied according to the experimental conditions in that it increased when the ATP concentration or temperature was decreased. Our results suggest that there is not a strict stoichiometric relationship between ATP hydrolysis and sliding distance in the dynein-tubulin system, indicating that the mechanochemical coupling is different from that in beating axonemes.