title: Single-cell quantitative RT-PCR analysis of Cpt1b and Cpt2 gene expression in mouse antral oocytes and in preimplantation embryos.
abstract: Fatty acids represent an important energy source for preimplantation embryos. Fatty acids oxidation is correlated with the embryo oxygen consumption which remains relatively constant up to the 8-cell stage, but suddenly increases between the 8-cell and morula stages. The degradation of fatty acids occurs in mitochondria and is catalyzed by several carnitine acyl transferases, including two carnitine palmitoyl transferases, CPT-I and CPT-II. We have carried out a study to determine the relative number of transcripts of Cpt1b and Cpt2 genes encoding for m-CPT-I and CPT-II enzymes, during mouse preimplantation development. Here we show that Cpt1b transcripts are first and temporally detected at the 2-cell stage and reappear at the morula and blastocyst stage. Cpt2 transcripts decrease following fertilization to undetectable levels and are present again later at the morula stage. These results show that transcription of both Cpt1b and Cpt2 is triggered at the morula stage, concomitantly with known increasing profiles of oxygen uptake and fatty acids oxidation. Based on the number of Cpt2 transcripts detected, we could discriminate the presence of two groups of embryos with high and low number of transcripts, from the zygote throughout preimplantation development. To further investigate if the establishment of these two groups of embryos occurs prior to fertilization, we have analyzed the relative number of transcripts of both genes in antral and ovulated MII oocytes. As for preimplantation embryos, MII oocytes show two groups of Cpt2 expression. Antral oocytes, classified according to their chromatin configuration in SN (surrounded nucleolus, in which the nucleolus is surrounded by a rim of Hoechst-positive chromatin) and NSN (not surrounded nucleolus, in which this rim is absent), show three groups with different numbers of Cpt2 transcripts. All NSN oocytes have a number of Cpt2 transcripts doubled compared to that of the group of MII oocytes with high expression. Instead, SN oocytes could be singled out into two groups with high and low numbers of Cpt2 transcripts, similar to those found in MII oocytes. The results of this study point out a correlation between the timing of fatty acids oxidation during preimplantation development and the expression of two genes encoding two enzymes involved in the oxidative pathway. Furthermore, although the biological meaning for the presence of two groups of oocytes/embryos with different levels of Cpt2 transcripts remains unclear, the data obtained suggest a possible correlation between the levels of Cpt2 expression and embryo developmental competence.
authors.affiliation: Laboratorio di Biologia dello Sviluppo e Centro di Eccellenza in Biologia Applicata, Dipartimento di Biologia Animale, Università degli Studi di Pavia, Pavia, Italy.
authors.forename: L
authors.initials: L
authors.lastname: Gentile
authors.forename: M
authors.initials: M
authors.lastname: Monti
authors.forename: V
authors.initials: V
authors.lastname: Sebastiano
authors.forename: V
authors.initials: V
authors.lastname: Merico
authors.forename: R
authors.initials: R
authors.lastname: Nicolai
authors.forename: M
authors.initials: M
authors.lastname: Calvani
authors.forename: S
authors.initials: S
authors.lastname: Garagna
authors.forename: C A
authors.initials: CA
authors.lastname: Redi
authors.forename: M
authors.initials: M
authors.lastname: Zuccotti
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000201:enzymology*; D002334:Carnitine O-Palmitoyltransferase / Q000235:genetics* / Q000502:physiology; D005227:Fatty Acids / Q000378:metabolism; D005260:Female; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D007041:Hypoxanthine Phosphoribosyltransferase / Q000096:biosynthesis; D007527:Isoenzymes / Q000235:genetics; D008297:Male; D008677:Metaphase; D051379:Mice; D009865:Oocytes / Q000166:cytology / Q000201:enzymology*; D010084:Oxidation-Reduction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012727:Sex Characteristics; D013094:Spermatozoa / Q000201:enzymology
chemical_list: D005227:Fatty Acids; D007527:Isoenzymes; D002334:Carnitine O-Palmitoyltransferase; D007041:Hypoxanthine Phosphoribosyltransferase