Conclusion. Taken together, paternal factors carried in ASG secretion affect genomic imprinting of developing embryos. The outcome of research work described here deepens our understanding of the role of ASG in maximizing reproductive performance mediated by regulating the epigenetic marks of the genome and in particular the imprinted genes. / Introduction. Our previous in vivo studies in golden hamster have shown the accessory sex glands (ASG) secretion facilitate the development of embryos to term but the underlying mechanism is still not clear. Since the deleterious effect caused by the lack of sperm exposure to ASG secretion is heritable to developing fetus and even after birth, we hypothesized that the paternal factor carried in ASG secretion may change the epigenetic regulation and in particular the imprinted genes of embryonic genome. / Materials and methods. Golden hamster and ICR mouse were used in this study. Hamster is a well-established animal model to study the effect of individual ASG but the genetic background of hamster is poorly known. To verify the specificity of our molecular probe and antibodies used in hamster, a mouse model was also established. Five groups of male hamsters and two groups of male mice were established by surgical treatment. In hamster, (SH) sham-operated, (VPX) ventral prostate-removed, (TX) all ASG-removed, (VPVX) castrated with ASG-removed except ventral prostate and (VX) castrated with intact ASG were established. In mouse, SH and VPX were established. In single-mating of hamster, male was copulated with female at estrus for 15 min. In double-mating of hamsters, female mated with each male for 10 min each. In single-mating of mouse, male was caged with female for 1 h. Epididymal sperm, uterine sperm, fertilized oocytes, pre-implantation embryos and fetuses at 13 days gestation (E13) were collected. Global DNA methylation of sperm, fertilized oocytes, early embryos and E13 fetuses were investigated by indirect immunofluorescence and DNA dot-blot using antibody against methylated DNA. Using the same technique, histone acetylation at lysine 5 residue was detected in male pronuclei of fertilized oocytes, protamine 1 and 2 content were detected in sperm, DNA methyltransferase 1, 3a and 3b activities were detected in early embryos. The crown-rump length and weight of fetuses were measured. Morphology was also examined under scanning electron microscope. Two sets of co-ordinately regulated but oppositely expressed imprinted genes Igf2/H19 and Dlk1/Gtl2 were investigated. H19 differentially methylated region (DMR) and Gtl2 promoter were examined by bisulfite sequencing in sperm and E13 fetuses. Expression of Igf2 and Dlk1 were examined by in situ hybridization and real-time PCR in pre-implantation embryos and E13 fetuses. / Results. Uterine sperm in VPX and TX groups showed no change of DNA methylation level and protamine 1 and 2 content. Fertilized oocytes in VPX and TX groups showed similar DNA methylation level as SH group in both hamster and mouse. Histone hypoacetylation was observed in male pronuclei of hamster but not in mouse. Early embryos in VPX and TX groups showed abnormal level of DNA methylation and Dnmt3b during embryo development in hamster. Replenishment of ASG secretion to sperm from VPX and TX group by double-mating restored the DNA methylation level to normal in early embryos. E13 fetuses of VPX and TX groups in hamster and VPX group in mouse showed DNA hypomethylation. E13 fetuses of VPX group in hamster showed increase in average crown-rump length and body weight with larger variations between individuals. One E13 fetus of VPX group in mouse showed polydactyly and malformation in the head. Real-time PCR showed abnormal expression of Igf2 and Dlk1 in both pre-implantation embryos and E13 fetuses of VPX and TX groups. Bisulfite sequencing showed hypermethylation of H19 DMR in VPX and TX groups of hamster and hypomethylation of Gtl2 promoter in VPX group of mouse. / "August 2007." / Adviser: Pak Ham Chow. / Source: Dissertation Abstracts International, Volume: 69-08, Section: B, page: 4739. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 194-224). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_344088 |
| Date | January 2007 |
| Contributors | Poon, Hong Kit., Chinese University of Hong Kong Graduate School. Division of Anatomy. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, theses |
| Format | electronic resource, microform, microfiche, 1 online resource (xxi, 240 p. : ill.) |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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