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The aim of this study was to determine the steady-state expression of 13 selected housekeeping genes in the myometrium of cyclic and pregnant cows. Cells taken from bovine myometrium on days 1-5, 6-10, 11-16 and 17-20 of the oestrous cycle and in weeks 3-5, 6-8 and 9-12 of pregnancy were used. Reverse transcribed RNA was amplified in real-time PCR using designed primers. Reaction efficiency was determined with the Linreg programme. The geNorm and NormFinder programmes were used to select the best housekeeping genes. They calculate the expression stability factor for each used housekeeping gene with the smallest value for most stably expressed genes. According to geNorm, the most stable housekeeping genes in the myometrium were C2orf29, TPB and TUBB2B, while the least stably expressed genes were 18S RNA, HPRT1 and GAPDH. NormFinder identified the best genes in the myometrium as C2orf29, MRPL12 and TBP, while the worst genes were 18S RNA, B2M and SF3A1. Differences in stability factors between the two programmes may also indicate that the physiological status of the female, e.g. pregnancy, affects the stability of expression of housekeeping genes. The different expression stability of housekeeping genes did not affect progesterone receptor expression but it could be important if small differences in gene expression were measured between studies.

Abstract. The aim of this study was to investigate the (1) expression of progesterone membrane component 1 (PGRMC1), serpine mRNA binding protein 1 (SERBP1) and progesterone receptor (PR) mRNA and (2) protein expression levels of PGRMC1, SERBP1 and PR isoforms A and B in the bovine myometrium during the estrous cycle and early pregnancy. Uteri from cows on days 1-5, 6-10, 11-16 and 17-21 of the estrous cycle and weeks 3-5, 6-8 and 9-12 of pregnancy were used (n=5-6 per period). There were no changes (P>0.05) in PGRMC1 mRNA expression during the estrous cycle, while expression of SERBP1 and PR mRNA was the lowest (P<0.05) on days 11-16 relative to other days of the cycle. The highest mRNA expression of PGRMC1, SERBP1 and PR was found during pregnancy. There were no changes (P>0.05) in SERBP1 protein expression in cycling and pregnant cows, while the highest (P<0.05) PGRMC1 protein expression was found during weeks 3-5 of pregnancy. Similar protein expression profiles for PRA and PRB were found, and protein levels were highest on days 1-5 of the estrous cycle. From day 6 of the cycle, PRA and PRB protein expression decreased and were maintained at this lower level during pregnancy. In conclusion, our study assessed mRNA and protein expression levels of PGRMC1, SERBP1 and PR in the bovine myometrium during the estrous cycle and the first trimester of pregnancy. It is possible that progesterone (P4) affects myometrial function in a genomic and nongenomic manner. P rogesterone (P4), which is produced in the corpus luteum (CL), regulates various female reproductive functions. This hormone is also responsible for morphological, functional and structural changes in the endometrium during the luteal phase and for suppression of myometrial contractions, which prepares the uterus for blastocyst implantation and ensures maintenance of pregnancy [1]. Many functions of P4 are mediated through binding to its specific nuclear progesterone receptor (nPR), which acts as a ligand-transcription factor and is expressed in two main isoforms, A (PRA) and B (PRB) [2]. PRA (94 kDa) is about 164 amino acids shorter than PRB (120 kDa) in humans [2], and a similar molecular mass was found in cattle [3,4]. The different isoforms of nPR play different roles in the cells. Isoform B acts mainly as an activator of progesterone-responsive genes, while PRA can inhibit the activity of PRB and other nuclear receptors such as the estrogen, glucocorticoid and mineralocorticoid receptors [2].P4 can also exert its effects more directly, by rapid, nongenomic pathways [5][6][7]. This nongenomic effect of P4 has been found in a number of tissues from the female reproductive tract [5,7,8] in mammals, including cows [9][10][11][12], but the nature of this mechanism is not fully understood. It has been suggested that P4, a lipophilic substance, can modify the fluidity of the cell membrane and thus change the affinity of membrane receptors for their ligands [9,13,14]. Moreover, P4 can be bound by its specific membrane receptor to stimulate early intracellular...

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