Identification of genes induced by the conceptus in the bovine endometrium during the pre-implantation period

Klein, Claudia (2006): Identification of genes induced by the conceptus in the bovine endometrium during the pre-implantation period. Dissertation, LMU München: Tierärztliche Fakultät

Abstract

An intact embryo-maternal communication in the pre-implantation period is particularly critical for establishment of pregnancy and early embryonic losses have been identified as the major cause of reproductive failure in cattle. Thus, to gain deeper insight into this complex embryo-maternal crosstalk, a combination of subtracted cDNA libraries and cDNA array hybridization was applied to identify mRNAs differentially regulated genes in the bovine endometrium by the presence of a conceptus. One cDNA library was constructed according the suppression subtractive hybridization method (Diatchenko et al., 1996) with minor modifications; a second cDNA library was constructed of subtracted cDNA purchased from the vertis Biotechnologie AG. As biological model endometrial tissue samples of monozygotic twins (generated by embryo splitting) collected at day 18 of gestation were used, which is a unique possibility to eliminate genetic variability as a factor potentially affecting the results of gene expression analyses. Array hybridization was carried out using 33P-labeled cDNA probes obtained from five monozygotic twin pairs. Sequence analysis revealed 87 different genes or mRNAs, respectively, which displayed a difference in signal intensity of 2.0 fold or more in at least four out of five twin pairs. Eighty genes corresponded to genes with known or inferred function, either the bovine gene or the human orthologue. For 7 mRNAs a match with bovine ESTs was obtained only. For nine selected genes the expression in the bovine endometrium was quantified by the use of quantitative real-time RT-PCR to verify the results obtained by array hybridization and to perform more precise quantitative measurements for these genes. Overall, the results of array hybridization and real-time RT-PCR correlated very well. Almost half of the identified genes are known to be stimulated by type I interferons reflecting the response to IFNt, which is the pregnancy recognition signal in ruminants. Of particular interest among the interferon stimulated genes is ISG15, one of the most markedly upregulated genes in the present study, which is hypothesized to stabilize intracellular endometrial proteins through conjugation processes. For the ISG15ylation system mRNAs of four potential components (IFITM1, IFITM3, HSXIAPAF1, and DTX3L) were found in addition to ISG15 and UBE1L, and in situ hybridization revealed similar mRNA expression patterns of these genes. It is therefore suggested, that modification of endometrial proteins through ISG15ylation plays a fundamental role in the IFNt signaling. A classification of the identified genes according to their assignment to Gene Ontologies revealed the orchestrated interaction of various processes and mechanisms with regard to the preparation of the maternal endometrium for embryonic implantation. As particular interesting, genes were identified involved in modulation of the maternal immune system at the humoral and cellular level, cell adhesion, cell communication, regulation of transcription, cell differentiation, cell growth, and cell proliferation. These findings underline that an intense embryo-maternal dialogue takes place during the pre-implantation period, which culminates in a receptive endometrium prepared for implantation of the conceptus. To conclude, this is the first study of its kind for cattle in the pre-implantation stage of embryonic development and revealed the orchestrated upregulation of genes important for embryonic implantation during the pre-implantation period in the bovine endometrium. The presented results provide new starting points for detailed investigations of the embryo-maternal dialogue by which the endometrium is prepared for conceptus attachment.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: Bos taurus, uterus, embryo-maternal crosstalk, SSH, microarray
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 590 Tiere (Zoologie)
• Fakultäten: Tierärztliche Fakultät
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 10. Februar 2006
• 1. Bericht­erstatter:in: Wolf, Eckhard