Teppei Goto

Epigenetic regulation of Kiss1 gene expression mediating estrogen-positive feedback action in the mouse brain

This study aims to determine the epigenetic mechanism regulating Kiss1 gene expression in the anteroventral periventricular nucleus (AVPV) to understand the mechanism underlying estrogen-positive feedback action on gonadotropin-releasing hormone/gonadotropin surge. We investigated estrogen regulation of the epigenetic status of the mouse AVPV Kiss1 gene locus in comparison with the arcuate nucleus (ARC), in which Kiss1 expression is down-regulated by estrogen. Histone of AVPV Kiss1 promoter region was highly acetylated, and estrogen receptor α was highly recruited at the region by estrogen. In contrast, the histone of ARC Kiss1 promoter region was deacetylated by estrogen. Inhibition of histone deacetylation up-regulated in vitro Kiss1 expression in a hypothalamic non–Kiss1-expressing cell line. Gene conformation analysis indicated that estrogen induced formation of a chromatin loop between Kiss1 promoter and the 3′ intergenic region, suggesting that the intergenic region serves to enhance estrogen-dependent Kiss1 expression in the AVPV. This notion was proved, because transgenic reporter mice with a complete Kiss1 locus sequence showed kisspeptin neuron-specific GFP expression in both the AVPV and ARC, but the deletion of the 3′ region resulted in greatly reduced GFP expression only in the AVPV. Taken together, these results demonstrate that estrogen induces recruitment of estrogen receptor α and histone acetylation in the Kiss1 promoter region of the AVPV and consequently enhances chromatin loop formation of Kiss1 promoter and Kiss1 gene enhancer, resulting in an increase in AVPV-specific Kiss1 gene expression. These results indicate that epigenetic regulation of the Kiss1 gene is involved in estrogen-positive feedback to generate the gonadotropin-releasing hormone/gonadotropin surge.

Interspecies organogenesis generates autologous functional islets

Islet transplantation is an established therapy for diabetes. We have previously shown that rat pancreata can be created from rat pluripotent stem cells (PSCs) in mice through interspecies blastocyst complementation. Although they were functional and composed of rat-derived cells, the resulting pancreata were of mouse size, rendering them insufficient for isolating the numbers of islets required to treat diabetes in a rat model. Here, by performing the reverse experiment, injecting mouse PSCs into Pdx-1-deficient rat blastocysts, we generated rat-sized pancreata composed of mouse-PSC-derived cells. Islets subsequently prepared from these mouse–rat chimaeric pancreata were transplanted into mice with streptozotocin-induced diabetes. The transplanted islets successfully normalized and maintained host blood glucose levels for over 370 days in the absence of immunosuppression (excluding the first 5 days after transplant). These data provide proof-of-principle evidence for the therapeutic potential of PSC-derived islets generated by blastocyst complementation in a xenogeneic host.

Lack of Pulse and Surge Modes and Glutamatergic Stimulation of Luteinising Hormone Release in Kiss1 Knockout Rats

Kisspeptin, encoded by the Kiss1 gene, has attracted attention as a key candidate neuropeptide in controlling puberty and reproduction via regulation of gonadotrophin‐releasing hormone (GnRH) secretion in mammals. Pioneer studies with Kiss1 or its cognate receptor Gpr54 knockout (KO) mice showed the indispensable role of kisspeptin‐GPR54 signalling in the control of animal reproduction, although detailed analyses of gonadotrophin secretion, especially pulsatile and surge‐mode of luteinising hormone (LH) secretion, were limited. Thus, in the present study, we have generated Kiss1 KO rats aiming to evaluate a key role of kisspeptin in governing reproduction via pulse and surge modes of GnRH/LH secretion. Kiss1 KO male and female rats showed a complete suppression of pulsatile LH secretion, which is responsible for folliculogenesis and spermatogenesis, and an absence of puberty and atrophic gonads. Kiss1 KO female rats showed no spontaneous LH/follicle‐stimulating hormone surge and an oestrogen‐induced LH surge, suggesting that the GnRH surge generation system, which is responsible for ovulation, does not function without kisspeptin. Furthermore, challenge of major stimulatory neurotransmitters, such as monosodium glutamate, NMDA and norepinephrine, failed to stimulate LH secretion in Kiss1 KO rats, albeit they stimulated LH release in wild‐type controls. Taken together, the results of the present study confirm that kisspeptin plays an indispensable role in generating two modes (pulse and surge) of GnRH/gonadotrophin secretion to regulate puberty onset and normal reproductive performance. In addition, the present study suggests that kisspeptin neurones play a critical role as a hub integrating major stimulatory neural inputs to GnRH neurones, using newly established Kiss1 KO rats, which serve as a useful model for detailed analysis of hormonal profiles.

Ability of tetraploid rat blastocysts to support fetal development after complementation with embryonic stem cells

This study was undertaken to generate rat offspring via tetraploid blastocyst complementation with embryonic stem (ES) cells. Tetraploid blastocysts were prepared by electrofusion of blastomeres from two‐cell stage embryos, and subsequent in vivo culture for 4 days. Microinjection into the tetraploid blastocoel of an inner cell mass isolated by immunosurgery resulted in the generation of rat offspring, suggesting the successful contribution of tetraploid blastocysts to their placenta. Tetraploid blastocyst complementation was attempted with a total of 4 ES cell lines (2 lines of female karyotype and 2 lines of male karyotype). In the rESWIv‐3i‐5 (XX) cell line, normal‐sized fetuses with heartbeats were harvested on E11.5 (12.1%), E12.5 (9.5%), and E13.5 (9.1%), but no viable fetuses were detected on E14.5. Similarly, use of the rESWIv‐3i‐1 (XX) cell line resulted in no viable fetus production on E14.5. Using the rESBLK2i‐1 (XY) cell line, viable fetuses were harvested not only on E11.5–E13.5 (2.6–5.5%), but also on E14.5 (3.0%). The transfer of a total of 487 tetraploid blastocysts complemented with rESBLK2i‐1 cells resulted in 256 implantation sites (52.6%) on E21.5, but no viable offspring was detected. Use of the rESBLK2i‐1/huKO (XY) cell line also resulted in no viable offspring production on E21.5. Analyses of the methylation pattern in differentially methylated regions and transcript level of genes that are imprinted in mice (H19, Meg3, Igf2r, Peg5, and Peg10) in the E14.5 conceptuses indicated a marked difference between the ES cell‐derived and control normal fetuses, but not between the tetraploid and control diploid placenta. Mol. Reprod. Dev. 79:402–412, 2012.

Molecular and Epigenetic Mechanism Regulating Hypothalamic Kiss1 Gene Expression in Mammals.

After the discovery of hypothalamic kisspeptin encoded by the Kiss1 gene, the central mechanism regulating gonadotropin-releasing hormone (GnRH) secretion, and hence gonadotropin secretion, is gradually being unraveled. This has increased our understanding of the central mechanism regulating puberty and subsequent reproductive performance in mammals. Recently, emerging evidence has indicated the molecular and epigenetic mechanism regulating hypothalamic Kiss1 gene expression. Here we compile data regarding DNA and histone modifications in the Kiss1 promoter region and provide a hypothetic scheme of the molecular and epigenetic mechanism regulating Kiss1 gene expression in two populations of hypothalamic kisspeptin neurons, which govern puberty and subsequent reproductive performance via GnRH/gonadotropin secretion.

Derivation of Embryonic Stem Cell Lines from Parthenogenetically Developing Rat Blastocysts

This study was undertaken to establish rat embryonic stem (ES) cells from parthenogenetically developing blastocysts. Ten blastocysts were prepared by treatment of ovulated rat oocytes with ionomycin and cycloheximide, and three alkaline phosphatase-positive ES cell lines were established using the N2B27 medium supplemented with mitogen activated protein kinase kinase inhibitor PD0325901, glycogen synthase kinase 3 inhibitor CHIR99021, rat leukemia inhibitory factor, and forskolin. Expression of stem cell marker genes (Oct-4, rNanog, Fgf-4, and Rex-1) was confirmed in all three ES cell lines by reverse transcriptase-polymerase chain reaction (RT-PCR). Combined bisulfite restriction analysis showed that the differentially methylated region locus of five imprinted genes (H19, Meg3IG, Igf2r, Peg5, and Peg10) in these ES cells remained to be demethylated or was hypomethylated, which was similar to that in control ES cells established from normal blastocysts. Characteristics of the parthenogenetic blastocyst-derived ES cells were successfully transmitted to the next generation through a chimeric rat for one of the three ES cell lines. This is the first report on germline-competent (genuine) ES cells derived from parthenogenetically developing rat blastocysts.

Pharmacological and Morphological Evidence of AMPK-Mediated Energy Sensing in the Lower Brain Stem Ependymocytes to Control Reproduction in Female Rodents

Ependymocytes are one of the energy-sensing cells that regulate animal reproduction through their responsiveness to changes in extracellular glucose levels and the expression of pancreatic-type glucokinase and glucose transporter 2, which play a critical role in sensing blood glucose levels in pancreatic β-cells. Molecular mechanisms underlying glucose sensing in the ependymocytes remain poorly understood. The AMP-activated protein kinase (AMPK), a serine/threonine kinase highly conserved in all eukaryotic cells, has been suggested to be an intracellular fuel gauge that detects cellular energy status. The present study aims to clarify the role AMPK of the lower brainstem ependymocytes has in sensing glucose levels to regulate reproductive functions. First, we will show that administration of 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside, an AMPK activator, into the 4th ventricle suppressed pulsatile LH release in female rats. Second, we will demonstrate the presence of AMPK catalytic subunit immunoreactivities in the rat lower brainstem ependymocytes. Third, transgenic mice were generated to visualize the ependymocytes with Venus, a green fluorescent protein, expressed under the control of the mouse vimentin promoter for further in vitro study. The Venus-labeled ependymocytes taken from the lower brainstem of transgenic mice revealed that AMPK activation by 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside, an AMPK activator, increased in vitro intracellular calcium concentrations. Taken together, malnutrition-induced AMPK activation of ependymocytes of the lower brainstem might be involved in suppression of GnRH/LH release and then gonadal activities.

Knock-in of a histone H2B-tdTomato reporter into the Rosa26 locus allows visualization of cell nuclei in rats

Fusion of histoneH2Bwith green fluorescent protein has made it possible to observe chromosomal dynamics in living mouse cells under a laser-scanning confocal microscope (Kanda et al., 1998), while knocking the tdTomato gene into the rat Rosa26 locus allows for ubiquitous transgene expression (Kobayashi et al., 2012). The present study merged these two approaches inserting a gene fusion of histone H2B and tdTomato into the rat Rosa26 locus via embryonic-stem-cell-mediated transgenesis so that cell nuclei could be visualized during rat preimplantation development. PCR products of the human H2B, tdTomato, splice acceptor sequence, and IRES-Neo-SV40pAwere inserted into theNheI site of prRosa26-1with an in-fusion cloning kit (Kobayashi et al., 2012). The finalH2B-tdTomato targeting vector was linearized by SalI digestion (Fig. S1A). The rBLK2i-1 embryonic stem cells (RGD ID: 10054010, http://rgd.mcw.edu/wg/home) were cultured on mitomycin C-treated mouse embryonic fibroblasts in N2B27 medium containing 1mM mitogen-activated kinase kinase (MEK) inhibitor PD0325901, 3mM glycogen synthase kinase 3 (GSK3) inhibitor CHIR99021, 1,000U/mL ESGRO 1 , and 10mM forskolin (Hirabayashi et al., 2014). Targeting vector (25mg) was introduced into 5 10 embryonic stem cells by electroporation at 800 V, 10mF in 500mL of N2B27 medium.Theelectroporated cellswere treatedwith 200mg/ mL G418 for 48 hr, resulting in 13 tdTomato-positive colonies seven days after the electroporation. Four out of the 13 embryonic stemcell linesweredetermined to carry a knockin at the Rosa26 locus (clones #1, 4, 5, 11; Fig. S1A) by PCR for tdTomato (primers: 50-GCGAGGAGGTCATCAAAGAG and 50-GATGACGGCCATGTTGTTGT) with AmpliTaq 1 DNA polymerase (Applied Biosystems, Foster City,CA), and for homologous recombination in theRosa26 locus (primers: 50-CAGAAAAGGCGGAGCGAGCCCAAG and 50-GGGCCCTCACATTGCCAAAAGACGG) with PrimeSTAR 1 GXL DNA polymerase (Takara Bio, Shiga, Japan). A total of 18 chimeric rats (female 7, male 11) were generated by injection of targeted embryonic stem cells (clone #1) into 64 Crlj:WI (RGD ID: 2312504) blastocysts, followed by transfer of the blastocysts to three pseudopregnant Crlj:WI recipients. Eighty-three F1 offspring were delivered from Crlj:WI females mated with six male chimeras (> 70% black-colored coat), and two out of eight black F1 rats were identified as H2B-tdTomato knock-in-positive by PCR for tdTomato. Normal viability and fertility of the homozygous knock-in rats demonstrated that this reporter is non-toxic and does not interfere with mitosis or meiosis. Parthenogenetic development of the established transgenic strain was observed from metaphase-II oocytes to blastocysts under an in vitro culture system (Fig. 1). Oocytes retrieved from superovulated, heterozygous knock-in females were denuded and cultured in mR1ECM medium after activation with ionomycin (5mM, 5min) followedbycycloheximideplus cytochalasinB (5mg/mLeach, 4 hr) treatment. Cell nuclei, including the meiotic plate and pronucleus, were successfully visualized by H2B-tdTomato expression during in vitro development. Thus, these H2B-tdTomato knock-in rats allow for the monitoring of chromosomal dynamics during embryonic development and may facilitate nuclear handling during rat cloning by somatic-cell nuclear transplantation.

Evidence of involvement of neurone‐glia/neurone‐neurone communications via gap junctions in synchronised activity of KNDy neurones

Pulsatile secretion of gonadotrophin‐releasing hormone (GnRH)/luteinising hormone is indispensable for the onset of puberty and reproductive activities at adulthood in mammalian species. A cohort of neurones expressing three neuropeptides, namely kisspeptin, encoded by the Kiss1 gene, neurokinin B (NKB) and dynorphin A, localised in the hypothalamic arcuate nucleus (ARC), so‐called KNDy neurones, comprises a putative intrinsic source of the GnRH pulse generator. Synchronous activity among KNDy neurones is considered to be required for pulsatile GnRH secretion. It has been reported that gap junctions play a key role in synchronising electrical activity in the central nervous system. Thus, we hypothesised that gap junctions are involved in the synchronised activities of KNDy neurones, which is induced by NKB‐NK3R signalling. We determined the role of NKB‐NK3R signalling in Ca2+ oscillation (an indicator of neuronal activities) of KNDy neurones and its synchronisation mechanism among KNDy neurones. Senktide, a selective agonist for NK3R, increased the frequency of Ca2+ oscillations in cultured Kiss1‐GFP cells collected from the mediobasal hypothalamus of the foetal Kiss1‐green fluorescent protein (GFP) mice. The senktide‐induced Ca2+ oscillations were synchronised in the Kiss1‐GFP and neighbouring glial cells. Confocal microscopy analysis of these cells, which have shown synchronised Ca2+ oscillations, revealed close contacts between Kiss1‐GFP cells, as well as between Kiss1‐GFP cells and glial cells. Dye coupling experiments suggest cell‐to‐cell communication through gap junctions between Kiss1‐GFP cells and neighbouring glial cells. Connexin‐26 and ‐37 mRNA were found in isolated ARC Kiss1 cells taken from adult female Kiss1‐GFP transgenic mice. Furthermore, 18β‐glycyrrhetinic acids and mefloquine, which are gap junction inhibitors, attenuated senktide‐induced Ca2+ oscillations in Kiss1‐GFP cells. Taken together, these results suggest that NKB‐NK3R signalling enhances synchronised activities among neighbouring KNDy neurones, and that both neurone‐neurone and neurone‐glia communications via gap junctions possibly contribute to synchronised activities among KNDy neurones.

Haploid embryonic stem cell lines derived from androgenetic and parthenogenetic rat blastocysts

The present study was conducted to establish haploid embryonic stem (ES) cell lines using fluorescent marker-carrying rats. In the first series, 7 ES cell lines were established from 26 androgenetic haploid blastocysts. However, only 1 ES cell line (ahES-2) was found to contain haploid cells (1n = 20 + X) by fluorescence-activated cell sorting (FACS) and karyotypic analyses. No chimeras were detected among the 10 fetuses and 41 offspring derived from blastocyst injection with the FACS-purified haploid cells. In the second series, 2 ES cell lines containing haploid cells (13% in phES-1 and 1% in phES-2) were established from 2 parthenogenetic haploid blastocysts. Only the phES-2 cell population was purified by repeated FACS to obtain 33% haploid cells. Following blastocyst injection with the FACS-purified haploid cells, no chimera was observed among the 11 fetuses; however, 1 chimeric male was found among the 47 offspring. Although haploid rat ES cell lines can be established from both blastocyst sources, FACS purification may be necessary for maintenance and chimera production.