RT-PCR specific errors in the quantification of mRNA transcript are easily generated by any variation in the amount of starting material between samples. These can happen through pipetting failures or by uncorrect photometric measurement of the total RNA amount per μl. The quantification errors are especially relevant when the samples have been obtained from different individuals like in this experiment, and can result in misinterpretation of the expression profiles of the target genes. Consequently, the question of what constitutes an appropriate standard arises (Thellin et al. 1999). The accepted method for minimising these errors and correcting for sample to sample variation is to amplify a cellular RNA that serves as an internal reference against which other RNA values can be normalised (Karge et al. 1998). The ideal standard should be expressed at a constant level among different tissues of an organism or in the same tissue of different individuals, at all stages of development, and should be unaffected by the experimental treatment. It was not possible to find such a housekeeping gene, which is stable expressed during oestrous cycle and induced luteolysis. Therefore we decided to calculate an index out of the four different housekeeping genes Histone, β-Actin, GAPDH and Ubiquitin using the Bestkeeper software (Pfaffl et al. 2004) and to normalise all target gene data with these index following the ΔΔCP method (Livak and Schmittgen 2001). There is evidence that the gene expression of all these four housekeeping genes changes during different physiological stages of the cells and that they have specific cell functions (Bustin 2000; Gillespie and Vousden 2003; Thellin et al. 1999; Warrington et al. 2000). Histones are abundant, basic, structural proteins that bind to the DNA and enable a tight packing of the DNA into nucleosomes. 147bp of DNA is folded over a Histone octamer (two molecules each of the four core Histones H2A, H2B, H3 and H4) to form a nucleosome. In between two nucleosomes another Histone (H1) bindes to 20bp of DNA to enable the spiralisation of the DNA into solenoides (Koolmann and Röhm 1998). Different variants were found for the four core Histones H2A, H2B, H3, H4 and for the linker Histone H1 (Gillespie and Vousden 2003; Pusarla and Bhargava 2005). These linker variant (H1.2) was found to be a pro-apoptotic factor in cells undergoing X-ray irridation (Konishi et al. 2003). These group showed that H1.2 has the ability to release cytochrom c from the mitochondria, which leads to apoptosis via the intrinsic pathway. How these release actually functions is not known definitively, but it seems to be a p53 dependent mechanism (Zong 2004). The pro-apoptotic action of H1.2 could be the reason for the
increase of mRNA expression of Histone from 12h to 64h shortly before the structural luteolysis begins. p53 shows nearly the same expression pattern like Histone, which could be due to its posssible regulatory action on Histone. β-Actin, a structur protein of the cytosceleton, is up-regulated during the first 7 days of the oestrous cycle and after 0.5h as well as from 24h to 64h after luteolysis. The luteal tissue of the developing CL consists of highly mitotic cells, mainly endothelial cells, which could explain the increase of expression at the early luteal stages. How β-Actin expression is regulated in apoptotic cells is presently not known. The work of Naora et al. (1995) indicates that β-Actin is differently expressed in different cells and that these expression is also dependend on the apoptotic stimulus. It is possible that PGF2α is responsible for the increased expression after 0.5h. At the time of structural luteolysis, 24h after PGF2α, apoptotic cells are found in the CL of marmoset monkeys (Young et al. 1998) showing the formation of apoptotic bodies. These formation effects the cytosceleton of the cells and is associated with the depolymerisation of Actin, which also plays a particular role in the formation and maintenance of these apoptotic bodies (Suarez-Huerta et al. 2000). These results might indicate the reason for the up-regulation of the β-Actin expression during structural luteolysis. GAPDH is an enzyme necessary for the glycolysis and gluconeogenesis. It shows an increased expression during the first seven days of the oestrous cycle, which could be due to the higher mitotic cell rate and the higher metabolic rate of these cells compared to the midluteal stage. It is reported that GAPDH expression is different during the cell cycle (Mansur et al. 1993) and that growth hormons can activate its transcription (Freyschuss et al. 1994). Two groups (Qi and Sit 2000b) also reported that GAPDH is up- regulated during apoptosis and that it is regulated by p53 (Chen et al. 1999). Our data show that GAPDH is not regulated during luteolysis, although an increase of expression can be seen from 24h onwards just like the expression of p53. It might be due to the greater variances in the groups that no significancies were found. Ubiquitin is necessary for the degradation of proteins and acts as regulator of all aspects of cell biology, including cell division, growth, communication, movement and apoptosis (Johnson 2002; Pickart 2001). It is reported that Ubiquitin is up-regulated during PGF2α induced luteolysis in marmoset monkeys (Young et al. 1998) and in sheep (Murdoch et al. 1996). This is in contrast to our data, which revealed a slight but significant decreased expression of Ubiquitin from 48h to 64h during induced luteolysis. The reason for these down-regulation is not know and has to be futher investigated. For the evaluation of factors in tissues undergoing apoptosis it seems to be necessary to normalise these datas with more than one HKG, because of their specific roles during apoptosis.
6.2.2 Extracellular matrix proteases in the CL during oestrous cycle and induced