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      DNA Methylation Patterns in the Hypothalamus of Female Pubertal Goats

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          Abstract

          Female pubertal development is tightly controlled by complex mechanisms, including neuroendocrine and epigenetic regulatory pathways. Specific gene expression patterns can be influenced by DNA methylation changes in the hypothalamus, which can in turn regulate timing of puberty onset. In order to understand the relationship between DNA methylation changes and gene expression patterns in the hypothalamus of pubertal goats, whole-genome bisulfite sequencing and RNA-sequencing analyses were carried out. There was a decline in DNA methylation levels in the hypothalamus during puberty and 268 differentially methylated regions (DMR) in the genome, with differential patterns in different gene regions. There were 1049 genes identified with distinct expression patterns. High levels of DNA methylation were detected in promoters, introns and 3′-untranslated regions (UTRs). Levels of methylation decreased gradually from promoters to 5′-UTRs and increased from 5′-UTRs to introns. Methylation density analysis demonstrated that methylation level variation was consistent with the density in the promoter, exon, intron, 5′-UTRs and 3′-UTRs. Analyses of CpG island (CGI) sites showed that the enriched gene contents were gene bodies, intergenic regions and introns, and these CGI sites were hypermethylated. Our study demonstrated that DNA methylation changes may influence gene expression profiles in the hypothalamus of goats during the onset of puberty, which may provide new insights into the mechanisms involved in pubertal onset.

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          Most cited references28

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          The non-coding Air RNA is required for silencing autosomal imprinted genes.

          In genomic imprinting, one of the two parental alleles of an autosomal gene is silenced epigenetically by a cis-acting mechanism. A bidirectional silencer for a 400-kilobase region that contains three imprinted, maternally expressed protein-coding genes (Igf2r/Slc22a2/Slc22a3) has been shown by targeted deletion to be located in a sequence of 3.7 kilobases, which also contains the promoter for the imprinted, paternally expressed non-coding Air RNA. Expression of Air is correlated with repression of all three genes on the paternal allele; however, Air RNA overlaps just one of these genes in an antisense orientation. Here we show, by inserting a polyadenylation signal that truncates 96% of the RNA transcript, that Air RNA is required for silencing. The truncated Air allele maintains imprinted expression and methylation of the Air promoter, but shows complete loss of silencing of the Igf2r/Slc22a2/Slc22a3 gene cluster on the paternal chromosome. Our results indicate that non-coding RNAs have an active role in genomic imprinting.
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            Regulation of Kiss1 gene expression in the brain of the female mouse.

            The Kiss1 gene encodes a family of neuropeptides called kisspeptins, which activate the receptor G protein-coupled receptor-54 and play a role in the neuroendocrine regulation of GnRH secretion. We examined whether estradiol (E2) regulates KiSS-1 in the forebrain of the female mouse by comparing KiSS-1 mRNA expression among groups of ovary-intact (diestrus), ovariectomized (OVX), and OVX plus E2-treated mice. In the arcuate nucleus (Arc), KiSS-1 expression increased after ovariectomy and decreased with E2 treatment. Conversely, in the anteroventral periventricular nucleus (AVPV), KiSS-1 expression was reduced after ovariectomy and increased with E2 treatment. To determine whether the effects of E2 on KiSS-1 are mediated through estrogen receptor (ER)alpha or ERbeta, we evaluated the effects of E2 in OVX mice that lacked functional ERalpha or ERbeta. In OVX mice that lacked functional ERalpha, KiSS-1 mRNA did not respond to E2 in either the Arc or AVPV, suggesting that ERalpha is essential for mediating the inhibitory and stimulatory effects of E2. In contrast, KiSS-1 mRNA in OVX mice that lacked functional ERbeta responded to E2 exactly as wild-type animals. Double-label in situ hybridization revealed that virtually all KiSS-1-expressing neurons in the Arc and AVPV coexpress ERalpha, suggesting that the effects of E2 are mediated directly through KiSS-1 neurons. We conclude that KiSS-1 neurons in the Arc, which are inhibited by E2, may play a role in the negative feedback regulation of GnRH secretion, whereas KiSS-1 neurons in the AVPV, which are stimulated by E2, may participate in the positive feedback regulation of GnRH secretion.
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              TAC3 and TACR3 mutations in familial hypogonadotropic hypogonadism reveal a key role for Neurokinin B in the central control of reproduction.

              The timely secretion of gonadal sex steroids is essential for the initiation of puberty, the postpubertal maintenance of secondary sexual characteristics and the normal perinatal development of male external genitalia. Normal gonadal steroid production requires the actions of the pituitary-derived gonadotropins, luteinizing hormone and follicle-stimulating hormone. We report four human pedigrees with severe congenital gonadotropin deficiency and pubertal failure in which all affected individuals are homozygous for loss-of-function mutations in TAC3 (encoding Neurokinin B) or its receptor TACR3 (encoding NK3R). Neurokinin B, a member of the substance P-related tachykinin family, is known to be highly expressed in hypothalamic neurons that also express kisspeptin, a recently identified regulator of gonadotropin-releasing hormone secretion. These findings implicate Neurokinin B as a critical central regulator of human gonadal function and suggest new approaches to the pharmacological control of human reproduction and sex hormone-related diseases.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, CA USA )
                1932-6203
                27 October 2016
                2016
                : 11
                : 10
                : e0165327
                Affiliations
                [1 ]Anhui Provincial Laboratory of Animal Genetic Resources Protection and Breeding, College of Animal Science and Technology, Anhui Agricultural University, 130 Changjiang West Road, Hefei, Anhui 230036, China
                [2 ]Anhui Provincial Laboratory for Local Livestock and Poultry Genetic Resource Conservation and Bio-Breeding, 130 Changjiang West Road, Hefei, Anhui 230036, China
                [3 ]Department of Animal Veterinary Science, College of Animal Science and Technology, Anhui Agricultural University, 130 Changjiang West Road, Hefei, Anhui 230036, China
                Harbin Institute of Technology, CHINA
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                • Conceptualization: CY JY FGF.

                • Data curation: XXG.

                • Formal analysis: YSL HGC.

                • Funding acquisition: FGF.

                • Investigation: HGC YHL.

                • Methodology: FGF XXG.

                • Project administration: FGF.

                • Resources: XRZ YL.

                • Software: XML KFZ LL.

                • Supervision: FGF.

                • Validation: JPD YHZ.

                • Visualization: FGF.

                • Writing – original draft: CY.

                • Writing – review & editing: FGF YL.

                Article
                PONE-D-16-28005
                10.1371/journal.pone.0165327
                5082945
                27788248
                b211f744-a1a1-42f5-a88b-083b90368875
                © 2016 Yang et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 13 July 2016
                : 10 October 2016
                Page count
                Figures: 4, Tables: 0, Pages: 12
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100001809, National Natural Science Foundation of China;
                Award ID: 31472096
                Funded by: funder-id http://dx.doi.org/10.13039/501100001809, National Natural Science Foundation of China;
                Award ID: 31301934
                Award Recipient :
                Funded by: National Transgenenic New Species Breeding Program of China
                Award ID: 2014ZX08008-005-004
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100003995, Natural Science Foundation of Anhui Province;
                Award ID: 1508085MC54
                The National Natural Science Foundation of China [grant number 31472096], the National Transgenenic New Species Breeding Program of China [grant number 2014ZX08008-005-004], the National Natural Science Foundation of China [grant number 31301934], and the Anhui Provincial Natural Science Foundation [grant number 1508085MC54].
                Categories
                Research Article
                Biology and life sciences
                Cell biology
                Chromosome biology
                Chromatin
                Chromatin modification
                DNA methylation
                Biology and life sciences
                Genetics
                Epigenetics
                Chromatin
                Chromatin modification
                DNA methylation
                Biology and life sciences
                Genetics
                Gene expression
                Chromatin
                Chromatin modification
                DNA methylation
                Biology and life sciences
                Genetics
                DNA
                DNA modification
                DNA methylation
                Biology and life sciences
                Biochemistry
                Nucleic acids
                DNA
                DNA modification
                DNA methylation
                Biology and life sciences
                Genetics
                Epigenetics
                DNA modification
                DNA methylation
                Biology and life sciences
                Genetics
                Gene expression
                DNA modification
                DNA methylation
                Medicine and Health Sciences
                Endocrinology
                Endocrine Physiology
                Puberty
                Biology and Life Sciences
                Physiology
                Endocrine Physiology
                Puberty
                Medicine and Health Sciences
                Physiology
                Endocrine Physiology
                Puberty
                Biology and Life Sciences
                Genetics
                Gene Expression
                Biology and Life Sciences
                Computational Biology
                Genome Complexity
                Introns
                Biology and Life Sciences
                Genetics
                Genomics
                Genome Complexity
                Introns
                Biology and Life Sciences
                Anatomy
                Brain
                Hypothalamus
                Medicine and Health Sciences
                Anatomy
                Brain
                Hypothalamus
                Biology and Life Sciences
                Organisms
                Animals
                Vertebrates
                Amniotes
                Mammals
                Ruminants
                Goats
                Biology and life sciences
                Molecular biology
                Molecular biology techniques
                Sequencing techniques
                RNA sequencing
                Research and analysis methods
                Molecular biology techniques
                Sequencing techniques
                RNA sequencing
                Biology and Life Sciences
                Computational Biology
                Genome Analysis
                Gene Ontologies
                Biology and Life Sciences
                Genetics
                Genomics
                Genome Analysis
                Gene Ontologies
                Custom metadata
                Data are available from the Sequence Read Archive (SRA) and the accession number is SRP077789.

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