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Epigenetic changes coincide with in vitro primate GnRH neuronal maturation.

ABSTRACT: Cellular and molecular mechanisms underlying pulsatile GnRH release are not well understood. In the present study, we examined the developmental changes in intracellular calcium dynamics, peptide release, gene expression, and DNA methylation in cultured GnRH neurons derived from the nasal placode of rhesus monkeys. We found that GnRH neurons were functionally immature, exhibiting little fluctuation in intracellular calcium ([Ca(2+)](i)) and sparse pulses of GnRH peptide release in the first 12 d in vitro (div). By 14-18 div, GnRH neurons exhibited periodic [Ca(2+)](i) oscillations, synchronizing at approximately 60-min intervals and GnRH pulses occurred at approximately 60-min intervals. Interestingly, the total GnRH peptide release further increased after 18 div. Measurement of GnRH mRNA and gene CpG methylation status at 0, 14, and 20 div indicated that mRNA levels significantly (P < 0.05) increased between 14 and 20 div, just as maximal decapeptide release was observed. By bisulfite sequencing across a 5' CpG island of the GnRH gene, we further found that methylation at eight of 14 CpG sites significantly (P < 0.05) decreased between 0 and 20 div. These data indicate that epigenetic differentiation occurs during GnRH neuronal development and suggest that increased GnRH gene expression and decreased CpG methylation status are molecular phenotypes of mature GnRH neurons. To our knowledge, this is the first report that developmental DNA demethylation occurs in postmitotic neurons toward a stable neuronal phenotype.

SUBMITTER: Kurian JR

PROVIDER: S-EPMC2954729 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

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Epigenetic changes coincide with in vitro primate GnRH neuronal maturation.

Kurian Joseph R JR Keen Kim L KL Terasawa Ei E

Endocrinology 20100922 11

Cellular and molecular mechanisms underlying pulsatile GnRH release are not well understood. In the present study, we examined the developmental changes in intracellular calcium dynamics, peptide release, gene expression, and DNA methylation in cultured GnRH neurons derived from the nasal placode of rhesus monkeys. We found that GnRH neurons were functionally immature, exhibiting little fluctuation in intracellular calcium ([Ca(2+)](i)) and sparse pulses of GnRH peptide release in the first 12 d ...[more]

PMID: 20861233

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Project description:BACKGROUND: Assisted reproductive technologies allow to utilize a limited number of fully grown oocytes despite the presence in the ovary of a large pool of meiotically incompetent gametes potentially able to produce live births. In vitro folliculogenesis could be useful to recruit these oocytes by promoting their growth and differentiation. METHODOLOGY/PRINCIPAL FINDINGS: In vitro folliculogenesis was performed starting from sheep preantral (PA) follicles to evaluate oocyte nuclear/epigenetic maturation. Chromatin configuration, quantification of global DNA methylation, and epigenetic remodelling enzymes were evaluated with immunocytochemistry, telomere elongation was assessed with the Q-FISH technique, while the DNA methylation status at the DMRs of maternally IGF2R and BEGAIN, and paternally H19 methylated imprinted genes was determined by bisulfite sequencing and COBRA. Specifically, 70% of PA underwent early antrum (EA) differentiation and supported in culture oocyte global DNA methylation, telomere elongation, TERT and Dnmt3a redistribution thus mimicking the physiological events that involve the oocyte during the transition from secondary to tertiary follicle. Dnmt1 anticipated cytoplasmic translocation in in vitro grown oocytes did not impair global and single gene DNA methylation. Indeed, the in vitro grown oocytes acquired a methylation profile of IGF2R and BEGAIN compatible with the follicle/oocyte stage reached, and maintained an unmethylated status of H19. In addition, the percentage of oocytes displaying a condensed chromatin configuration resulted lower in in vitro grown oocytes, however, their ability to undergo meiosis and early embryo development after IVF and parthenogenetic activation was similar to that recorded in EA follicle in vivo grown oocytes. CONCLUSIONS/SIGNIFICANCE: In conclusion, the in vitro folliculogenesis was able to support the intracellular/nuclear mechanisms leading the oocytes to acquire a meiotic and developmental competence. Thus, the in vitro culture may increase the availability of fertilizable oocytes in sheep, and become an in vitro translational model to investigate the mechanisms governing nuclear/epigenetic oocyte maturation.

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Epigenetic changes coincide with in vitro primate GnRH neuronal maturation.

Publications

Epigenetic changes coincide with in vitro primate GnRH neuronal maturation.

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