A 270-base pair (bp) cDNA product was amplified by RT-PCR. The PCR products from 8 animals (3 preluteolytic and 5 postluteolytic) are shown in Figure 1a. The identity of the band was confirmed by DNA sequencing and was 100% homologous with the expected cDNA sequence described by Wempe et al.. The intensity of the amplified cDNA product, measured after ethidium bromide staining of agarose gels, was significantly greater (p < 0.05) in luteal tissue from animals that had already undergone functional luteolysis (n = 5) compared to the other animals in the group (n = 9) that still had a functional CL (Fig. 2). There was no difference in the intensity of the amplified band obtained with the ATPase oligomers in CL collected before and after the onset of structural luteolysis (Fig. 1b). ampicillin antibiotic
In Situ Hybridization
In situ hybridization was performed on luteal tissue from a subset of animals before (n = 3) and after (n = 3) the onset of functional luteolysis. Representative in situ results for both groups of animals are presented in Figure 3. The intensity of the signal (the number of graphic pixels containing a silver grain) was significantly greater (p < 0.05) in CL from cows after functional luteolysis compared to CL collected before luteolysis (Table 1).
Table 1. MCP-1 mRNA expression (mean ± SEM) in the bovine CL measured by in situ hybridization.a
|Cow||Day of cycle||Progesterone(ng/ml)||PGFM(pg/ml)||mRNA expression13|
|983c||19||3.6||114||0.31 ± 0.04|
|958c||17||4.3||81||0.32 ± 0.09|
|982d||19||0.7||232||9.9 ± 2.6|
|984d||20||0.7||117||16.5 ± 3.7|
|955d||19||0.9||112||18.6 ± 4.6|
a Systemic progesterone (ng/ml) and PGFM concentrations (pg/ml) were measured at the time of tissue collection. b Percentage pixels occupied by silver grains. c Preluteolytic corpus luteum. d Postluteolytic corpus luteum.