Magdalena K. Kowalik

The putative roles of nuclear and membrane-bound progesterone receptors in the female reproductive tract☆

Progesterone produced by the corpus luteum (CL) is a key regulator of normal cyclical reproductive functions in the females of mammalian species. The physiological effects of progesterone are mediated by the canonical genomic pathway after binding of progesterone to its specific nuclear progesterone receptor (PGR), which acts as a ligand-activated transcription factor and has two main isoforms, PGRA and PGRB. These PGR isoforms play different roles in the cell; PGRB acts as an activator of progesterone-responsive genes, while PGRA can inhibit the activity of PGRB. The ratio of these isoforms changes during the estrous cycle and pregnancy, and it corresponds to the different levels of progesterone signaling occurring in the reproductive tract. Progesterone exerts its effects on cells also by a non-genomic mechanism by the interaction with the progesterone-binding membrane proteins including the progesterone membrane component (PGRMC) 1 and 2, and the membrane progestin receptors (mPRs). These receptors rapidly activate the appropriate intracellular signal transduction pathways, and subsequently they can initiate specific cell responses or modulate genomic cell responses. The diversity of progesterone receptors and their cellular actions enhances the role of progesterone as a factor regulating the function of the reproductive system and other organs. This paper deals with the possible involvement of nuclear and membrane-bound progesterone receptors in the function of target cells within the female reproductive tract.

Expression of progesterone receptor membrane component (PGRMC) 1 and 2, serpine mRNA binding protein 1 (SERBP1) and nuclear progesterone receptor (PGR) in the bovine endometrium during the estrous cycle and the first trimester of pregnancy

Progesterone (P4) is involved in the regulation of essential reproductive functions affecting the target cells through both nuclear progesterone receptors (PGRs) and membrane progesterone receptors. The aim of this study was to determine the mRNA and protein expression for PGRMC1, PGRMC2, SERBP1 and PGR within the bovine endometrium during the estrous cycle and the first trimester of pregnancy. There were no changes in PGRMC1 and PGRMC2 mRNA and protein expression during the estrous cycle, however, mRNA levels of PGRMC1 and PGRMC2 were increased (P<0.001) in pregnant animals. SERBP1 mRNA expression was increased (P<0.05), while the level of this protein was decreased (P<0.05) on days 11-16 of the estrous cycle. The expression of PGR mRNA was higher (P<0.01) on days 17-20 compared to days 6-10 and 11-16 of the estrous cycle and pregnancy. PGR-A and PGR-B protein levels were elevated on days 1-5 and 17-20 of the estrous cycle as compared to other stages of the cycle and during pregnancy. In conclusion, our results indicate that P4 may influence endometrial cells through both genomic and nongenomic way. This mechanism may contribute to the regulation of the estrous cycle and provide protection during pregnancy.

Progesterone receptor membrane component 1 (PGRMC1) gene expression in corpus luteum during the estrous cycle in cows.

The aim of the study was to investigate progesterone receptor membrane component 1 (PGRMC1) mRNA expression in bovine corpus luteum (CL) obtained from heifers or non-pregnant cows on the following days of the estrous cycle: 1-5, 6-10, 11-16 and 17-21 (n=4/each time period). The expression of PGRMC1 mRNA, analyzed by semiquantitative RT-PCR, was the highest on days 6-10 (p<0.01) and then it declined (p<0.05). The lowest expression was found on days 1-5 (p<0.05). A significant correlation (p<0.05) was also observed between luteal progesterone (P(4)) concentration and PGRMC1 mRNA expression. These data indicate that PGRMC1 mRNA is expressed in bovine CL and this expression varies throughout the luteal phase. It is assumed that PGRMC1 may be involved in a non-genomic effect of P(4) on luteal cells.

The effect of ovarian steroids on oxytocin-stimulated secretion and synthesis of prostaglandins in bovine myometrial cells

The aim was to study whether bovine myometrial cells have an enzymatic system able to produce prostaglandins (PGs) and whether PGs synthesis is regulated by steroids in a similar manner as endometrial cells. In Experiment 1, immunohistochemical studies localized proteins for cyclooxygenase 2 (COX2), PGE synthase (PGES) and PGF2alpha synthase (PGFS) in myometrial and endometrial (as positive control) slices from days 14 to 16 of the estrous cycle. Enzymatically isolated myometrial cells (2.5 x 10(5)/ml) were cultured for 96 h to attach them to the bottom of the culture well. In Experiment 2, cells were preincubated for 30 min with progesterone (P4; 10(-5) M), and thereafter incubated for 4 or 6h with arachidonic acid (AA; 10(-5) M, as positive control), oxytocin (OT; 10(-7) M), and OT+P4. In medium, PGE and PGFM (PGF2alpha metabolite) were increased (P<0.05) by AA treatment after 4 and 6h, but by OT only after 6h of incubation. Progesterone did not affect (P>0.05) basal secretion of both PGs, but it diminished (P<0.05) the stimulatory effect of OT on PGF2alpha and PGE secretion after 6h of incubation. The amount of enzyme protein for COX2, PGES and PGFS analyzed by Western Blot was affected (P>0.05) by any of the factors added to the culture medium. In Experiment 3, myometrial cells were preincubated with P4 (10(-5)M) and pregnenolone (P5; 10(-5)M) for 30 min, and then incubated for 6h with OT (10(-7) M) and OT plus each of these steroids used. Expression of mRNA for COX2, but not PGFS and PGES, was found in the cells stimulated with OT. Neither P4 nor P5 affected expression of the studied genes; however, both steroids diminished (P<0.05) OT-stimulated mRNA expression of COX2. The data suggest that: (a) myometrial cells are able to synthesize both PGF2alpha and PGE and (b) synthesis of these PGs may be regulated by steroids through a transcription-independent manner, which modulated the effect of OT on COX2 mRNA expression.

Cloning and expression of progesterone receptor isoforms A and B in bovine corpus luteum.

Progesterone (P4) affects a cell through its nuclear receptor (PGR), which has two main isoforms: A (PGRA) and B (PGRB). A partial section of previously unknown PGRB cDNA from cattle was cloned. Next, mRNA and protein levels for these two isoforms in corpora lutea (CL) collected during different stages of the oestrous cycle and pregnancy were determined. The PGRB mRNA level was highest on Days 2-5 of the oestrous cycle, decreased over the next few days (P<0.01) and increased again slightly on Days 17-20 (P<0.05). During pregnancy, PGRB mRNA was at its lowest level during Weeks 3-5 (P<0.01) and highest during Weeks 6-12 (P<0.01). The profile of PGRA mRNA levels was similar to that of PGRB throughout the oestrous cycle. The PGRA protein level was highest on Days 2-10 of the oestrous cycle, decreased continuously to its lowest concentration on Days 17-20 (P<0.01) and during Weeks 3-5 of pregnancy (P>0.05) and increased during Weeks 6-12 (P<0.05). PGRB protein concentration followed a similar pattern but at a markedly lower level. Both PGRA and PGRB isoforms are involved in the regulation of P4 action, especially in the newly formed CL and developed CL in the first trimester of pregnancy. These data suggest that the variable expression of these isoforms during the oestrous cycle may depend on the influence of P4.

Validation of housekeeping genes for studying differential gene expression in the bovine myometrium

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.

Expression and immunolocalization of membrane progesterone receptors in the bovine oviduct

The oviduct plays a crucial role in the transport and maturation of gametes and ensures suitable conditions for fertility and early embryo development. One regulator of oviduct function is progesterone (P4), which affects the cell by interacting with nuclear progesterone receptors (PGRs) and through nongenomic mechanisms, presumably involving membrane PGRs. The aim of this study was to evaluate the expression of messenger RNAS (mRNAs) and proteins for progesterone receptor membrane component (PGRMC) 1 and 2 and membrane progestin receptors (mPR) α, β, and γ and to use immunohistochemistry to demonstrate their cell-specific localization in the bovine oviduct. Oviducts ipsilateral and contralateral to the corpus luteum or to the dominant follicle were collected from cows on days 6 to 12 (midluteal stage) and 18 to 20 (follicular stage) of the estrous cycle and divided into 3 parts (infundibulum, ampulla, and isthmus). There were no differences (P > 0.05) in the PGRMC1, PGRMC2, mPRα, β, and γ mRNA expression between ipsi- and contralateral oviducts. However, the same parts of the oviduct collected during the different stages of the estrous cycle showed higher (P < 0.05) mRNA levels of PGRMC1, PGRMC2, and mPRα on days 18 to 20 than on days 6 to 12 of the estrous cycle. mPRα and mPRβ mRNA levels were higher (P < 0.05) in the infundibulum than in the isthmus, whereas PGRMC1 expression was higher (P < 0.05) in the infundibulum than in ampulla. Immunohistochemistry was used to detect PGRMC1, PGRMC2, PRα, β, and γ proteins in all parts of both oviducts from days 6 to 12 and 18 to 20 of the estrous cycle. There were no differences in the staining intensity and cellular localization of the studied proteins between the ipsi- and contralateral oviducts and between the studied stages of the estrous cycle. A strong positive reaction was observed in luminal cells, but this reaction was less evident in myocytes and stromal cells. All proteins were also localized to the endothelial cells of blood vessels. These results suggest that membrane progesterone receptors, may be involved in the regulation of oviduct motility, secretory function, and blood flow in this organ.

Luteotropic and luteolytic factors regulate mRNA and protein expression of progesterone receptor isoforms A and B in the bovine endometrium

The aim of the present study was to examine the effects of luteotropic and luteolytic factors on the mRNA and protein levels of progesterone receptor isoforms A (PGRA) and B (PGRB) in the bovine endometrium. Endometrial slices from Days 6-10 and 17-20 of the oestrous cycle were treated with LH (100ngmL-1), oestradiol (E2; 1×10-8M), prostaglandin (PG) E2 (1×10-6M) and PGF2α (1×10-6M) and the nitric oxide donor NONOate (1×10-4M); these treatments lasted for 6h for mRNA expression analysis and 24h for protein expression analysis. On Days 6-10 of the oestrous cycle PGRAB (PGRAB; the entire PGRA mRNA sequence is common to the PGRB mRNA sequence) mRNA expression in endometrial slices was enhanced by E2 treatment (P<0.001), whereas PGRB mRNA expression was increased by LH (P<0.001), E2 (P<0.05) and NONOate (P<0.05) treatment. On Days 17-20, PGRAB mRNA expression increased after E2 (P<0.001) and PGE2 (P<0.05) treatment; PGRB mRNA expression was increased by PGE2 (P<0.05) and PGF2α (P<0.01) treatment, but decreased by LH (P<0.05). On Days 6-10 protein levels of PGRA were stimulated by E2 (P<0.01), whereas PGRB protein levels were increased by LH (P<0.05) and E2 (P<0.05). On Days 17-20 of the oestrous cycle, PGRA protein levels were enhanced by E2 (P<0.05) and PGF2α (P<0.05), whereas PGRB protein levels were stimulated by PGE2 (P<0.05) and PGF2α (P<0.001). These data suggest that luteotropic and luteolytic factors affect PGRA and PGRB mRNA and protein levels, and this may regulate the effects of progesterone on endometrial cells.

Expression and localization of progesterone receptor membrane component 1 and 2 and serpine mRNA binding protein 1 in the bovine corpus luteum during the estrous cycle and the first trimester of pregnancy.

The aim of this study was to evaluate the mRNA and protein expression and the localization of progesterone receptor membrane component 1 (PGRMC1), PGRMC2, and the PGRMC1 partner serpine mRNA binding protein 1 (SERBP1) in the bovine CL on Days 2 to 5, 6 to 10, 11 to 16, and 17 to 20 of the estrous cycle as well as during Weeks 3 to 5, 6 to 8, and 9 to 12 of pregnancy (n = 5-6 per each period). The highest levels of PGRMC1 and PGRMC2 mRNA expression were found on Days 6 to 16 (P < 0.05) and 11 to 16, respectively, of the estrous cycle and during pregnancy (P < 0.001). The level of PGRMC1 protein was the highest (P < 0.05) on Days 11 to 16 of the estrous cycle compared with the other stages of the estrous cycle and pregnancy, whereas PGRMC2 protein expression (P < 0.001) was the highest on Days 17 to 20 and also during pregnancy. The mRNA expression of SERBP1 was increased (P < 0.05) on Days 11 to 16, whereas the level of its protein product was decreased (P < 0.05) on Days 6 to 10 of the estrous cycle and was at its lowest (P < 0.001) on Days 17 to 20. In pregnant cows, the patterns of SERBP1 mRNA and protein expression remained constant and were comparable with those observed during the estrous cycle. Progesterone receptor membrane component 1 and PGRMC2 localized to both large and small luteal cells, whereas SERBP1 was observed mainly in small luteal cells and much less frequently in large luteal cells. All proteins were also localized in the endothelial cells of blood vessels. The data obtained indicate the variable expression of PGRMC1, PGRMC2, and SERBP1 mRNA and protein in the bovine CL and suggest that progesterone may regulate CL function via its membrane receptors during both the estrous cycle and pregnancy.

Onapristone (ZK299) and mifepristone (RU486) regulate the messenger RNA and protein expression levels of the progesterone receptor isoforms A and B in the bovine endometrium

The aim of this study was to examine whether progesterone (P(4)) and its antagonists, onapristone (ZK299) and mifepristone (RU486), affect the levels of PGRA and PGRB messenger RNA (mRNA) and protein in the cow uterus which may be important in understanding whether the final physiological effect evoked by an antagonist depends on PGR isoform bound to the antagonist. Endometrial slices on Days 6 to 10 and 17 to 20 of the estrous cycle were treated for 6 or 24 hours for mRNA and protein expression analysis, respectively, with P4, ZK299, or RU486 at a dose of 10(-4), 10(-5), or 10(-6) M. In the samples on Days 6 to 10 of the estrous cycle, PGRAB mRNA was stimulated by P(4) (10(-4) M; P < 0.01) and RU486 (10(-6); P < 0.001) and was decreased by ZK299 (10(-5); P < 0.05). In contrast, PGRB mRNA was decreased by the all P(4) (P < 0.01) and ZK299 (P < 0.001) doses and by two of the RU486 doses (10(-4) M; P < 0.01 and 10(-5) M; P < 0.01). In samples on Days 17 to 20 of the estrous cycle, PGRAB mRNA was stimulated by RU486 (10(-5) M; P < 0.001). PGRB mRNA was decreased by P(4) (10(-4) and 10(-5) M; P < 0.001), ZK299 (10(-4) and 10(-5) M; P < 0.001), and RU486 (10(-4) M; P < 0.01 and 10(-6) M; P < 0.001) and was increased by ZK299 (10(-6) M; P < 0.001) and RU486 (10(-5) M; P < 0.001). In samples on Days 6 to 10 of the estrous cycle, PGRB protein levels were decreased (P < 0.05) by all three ZK299 doses and by two of the RU486 doses (10(-4) M; P < 0.05 and 10(-5) M; P < 0.01). In contrast, in samples on Days 17 to 20, both PGRA and PGRB protein levels were decreased by ZK299 stimulation (10(-5) M; P < 0.05 and 10(-5) M; P < 0.01, respectively), whereas only PGRA protein levels were increased by RU486 (10(-5) M; P < 0.01). Both ZK299 and RU486 may exhibit both agonist and antagonist properties depending on which receptor isoform they affect. As a result, an increase or decrease in the expression of a particular PGR isoform will be observed.