Effects of commonly used carbamates (carbaryl and thiram) on the regulatory, secretory and motor functions of bovine cervixes in vitro.

Previously studied classes of pesticides, including organochlorines, organophosphates and pyrethroids disturb the mechanism that causes bovine myometrial contractions. Hence, the aim of this study was to investigate the effects of carbaryl and thiram, which are representative carbamate pesticides commonly used in global agriculture, on the motor and secretory functions of bovine cervixes. Additionally, the impacts of these pesticides on intra- and intercellular signaling in vitro were estimated. In this study, cervical cells or strips were obtained from cows at days 18-20 of the estrous cycle and were treated with carbaryl or thiram. Neither carbamate (10 or 100 ng/ml) exerted cytotoxic effects. Carbaryl increased the level of mRNA (at a dose of 0.1 ng/ml) and protein (at both doses, 1 and 10 ng/ml) expression for the oxytocin receptor (OXTR), while thiram (at 0.1 and 10 ng/ml or 0.1-10 ng/ml, respectively) caused the opposite effects. Moreover, the level of the second messenger inositol-trisphosphate (IP3) was decreased by carbaryl (10 ng/ml) but increased by thiram (10 ng/ml). Only thiram decreased prostaglandin-endoperoxide synthase 2 (PTGS2; 0.1 ng/ml) and aldo-keto reductase family 1, member B1 (AKR1B1; 0.1 ng/ml), and prostaglandin E synthase 2 (PTGES2; 0.1-10 ng/ml) mRNA expression, while thiram (0.1-10 ng/ml) and carbaryl (0.1 and 10 ng/ml) both decreased the release of PGF2α. Carbaryl (10 ng/ml) and thiram (10 ng/ml) also decreased the level of a gap junction protein (GAP). Moreover, carbaryl (10 ng/ml) decreased the level of myosin light chain kinase (MLCK). However, the strength of cervical contractions was increased by thiram (1 and 10 ng/ml) but decreased by carbaryl (1 and 10 ng/ml). Carbaryl increased the receptivity of cervical cells to oxytocin (OXT), but inhibited further transduction (IP3) of this signal. Hence, direct inhibition of cervical strip contraction may occur. In contrast, thiram mostly decreased the receptivity of cervical cells to OXT, while it stimulated the contraction of cervical strips. Moreover, compared to carbaryl, thiram more greatly affected the synthesis and release of prostaglandins. These results suggest that carbaryl and thiram disturb OXT signaling, PG secretion and cervical contraction in vitro.

Luteotropic and Luteolytic Factors Modulate the Expression of Nuclear Receptor Coregulators in Bovine Luteal Cells Independently of Histone Acetyltransferase and Histone Deacetylase Activities.

The aims of this study were to examine the effect of luteotropic and luteolytic factors on the mRNA and protein expression of the coactivators HAT: histone acetyltransferase p300 (P300), cyclic adenosine monophosphate response element-binding protein (CREB), and steroid receptor coactivator-1 (SRC-1) and the corepressor: nuclear receptor corepressor-2 (NCOR-2) in bovine luteal cells on days 6-10 and 16-20. HAT and HDAC activities were also measured. The obtained results showed that luteotropic and luteolytic factors influence changes in the mRNA and protein levels of the coregulators of PGRs. However, they did not affect the activity of related HAT and HDAC, respectively. Therefore, it is possible that these factors, through changes in the expression of nuclear receptor coactivators and corepressors, may affect the functioning of the nuclear receptors, including PGRs, in the bovine CL.

Effect of Steroid Hormones, Prostaglandins (E2 and F2α), Oxytocin, and Tumor Necrosis Factor Alpha on Membrane Progesterone (P4) Receptors Gene Expression in Bovine Myometrial Cells.

Myometrium tissue shows the expression of non-genomic membrane progesterone (P4) receptors, such as progesterone receptor membrane components (PGRMC) 1 and 2 and membrane progestin receptors (mPR) alpha (mPRα), beta (mPRß), and gamma (mPRγ). Their variable expression in the bovine uterus during the estrous cycle and early pregnancy suggests that ovarian steroids and luteotropic and/or luteolytic factors may regulate the expression of these receptors in the myometrium. Therefore, this study aimed to examine the effect of P4, estradiol (E2), P4 with E2, prostaglandins (PG) E2 and F2α, oxytocin (OT), and tumor necrosis factor α (TNFα) on the gene expression of PGRMC1, PGRMC2, serpine-1 mRNA-binding protein (SERBP1), and mPRα, mPRß, and mPRγ in bovine myometrial cells from days 6 to 10 and 11 to 16 of the estrous cycle. The PGE2 concentration and mRNA expression were determined by EIA and real-time PCR, respectively. The data indicated that P4 and E2 can affect the mRNA expression of all studied receptors and SERPB1. However, PGE2, OT, and TNFα could only modulate the expression of PGRMC1, PGRMC2, and SERPB1, respectively. Steroids/factors changed the expression of PGRMC and mPR genes depending on the dose, the stage of the estrous cycle, and the types of receptors. This suggests that the local hormonal milieu may influence the activity of these receptors and P4 action in myometrial cells during the estrous cycle.

Do commonly used herbicides (atrazine and glyphosate) have the potential to impair the contractions, prostaglandin releasing and conducting of oxytocin signal at the bovine cervix in vitro?

Glyphosate (Gly) and atrazine (Atr) are among the most commonly used herbicides in global agriculture. It was previously shown that both Atr and Gly impair the ovarian and uterine secretion of regulators of myometrial motility (oxytocin (OT) or prostaglandins (PGs)) in cows, and Atr can also decrease the force of contractions in strips from the uterine horn. Hence, the aim of this study was to compare the effects of Atr and Gly on the motor and secretory function of the bovine cervix in vitro as well as receptivity and signal transduction in cervical cell cultures. Cervical strips or cells obtained from cows before ovulation were treated with environmental doses of Atr or Gly (0.1-10 ng/ml) since these herbicides exerted no cytotoxic effect at a dose of 100 ng/ml. Only Atr increased the force of cervical contractions, while both Atr and Gly decreased the secretion of prostaglandins (PGs) without disturbing their synthesis. Moreover, Atr decreased the mRNA expression and protein level of oxytocin receptor (OTR), while Gly increased OTR protein levels. Both Atr and Gly decreased the contents of gap junction proteins (GAPs), Atr decreased the contents of second messengers (diacylglycerol - DAG, inositol-tris-phosphate - IP3), and Gly decreased the level of myosin light chain kinase (MLCK) but increased DAG levels. Atr directly enhanced the cervical strips contractions. Both herbicides disturbed cellular signalling and inhibited PGs secretion. It suggest that Atr and Gly have the potential to impair the activity of cervical cells in vitro, which might be followed by failure of maintenance with gestation.

Steroid Receptor Coregulators Can Modulate the Action of Progesterone Receptor during the Estrous Cycle in Cow Endometrium.

Nuclear receptor coregulators include coactivators and corepressors which associate with the progesterone receptor (PGR) during its activation. Fluctuations in the transcription levels of their respective genes and subsequent protein production as well as in related activities for histone acetyltransferase (HAT) and histone deacetylase (HDAC) can affect PGR function and thus change the action of progesterone (P4) in bovine endometrium during the estrous cycle. Endometrial tissue on days 2-5, 6-10, 11-16, and 17-20 of the estrous cycle was used for determination of the mRNA expression levels of coactivators P300, CREB, and SRC-1 along with corepressor NCOR-2 using Real-Time PCR, with protein levels by Western blot. Coregulators cellular localizations were assessed by immunohistochemistry whereas the activities of HAT and HDAC by using EIA. The highest levels of mRNA and proteins for all of the investigated coregulators, as well as the highest levels of activity for HAT and HDAC, were detected over days 2-16 of the estrous cycle. All of the tested coregulatory proteins were localized in the nuclei of endometrial cells. This research indicates the important role of coregulators of the PGR receptor in regulating P4 activity in endometrial cells, especially during the pre-implantation period.

Progesterone Receptor Coregulators as Factors Supporting the Function of the Corpus Luteum in Cows.

Progesterone receptor (PGR) for its action required connection of the coregulatory proteins, including coactivators and corepressors. The former group exhibits a histone acetyltransferase (HAT) activity, while the latter cooperates with histone deacetylase (HDAC). Regulations of the coregulators mRNA and protein and HAT and HDAC activity can have an indirect effect on the PGR function and thus progesterone (P4) action on target cells. The highest mRNA expression levels for the coactivators-histone acetyltransferase p300 (P300), cAMP response element-binding protein (CREB), and steroid receptor coactivator-1 (SRC-1)-and nuclear receptor corepressor-2 (NCOR-2) were found in the corpus luteum (CL) on days 6 to 16 of the estrous cycle. The CREB protein level was higher on days 2-10, whereas SRC-1 and NCOR-2 were higher on days 2-5. The activity of HAT and HDAC was higher on days 6-10 of the estrous cycle. All of the coregulators were localized in the nuclei of small and large luteal cells. The mRNA and protein expression levels of the examined coactivators and corepressor changed with the P4 level. Thus, P4 may regulate CL function via the expression of coregulators, which probably affects the activity of the PGR.

Expression of membrane progestin receptors (mPRs) α, ß and γ in the bovine uterus during the oestrous cycle and pregnancy.

Progesterone (P4) affects cell function through the nuclear progesterone receptor and membrane-bound progesterone binding proteins, including the membrane progestin receptors (mPRs) alpha (mPRα), beta (mPRß) and gamma (mPRγ), which belong to the progestin and adipoQ receptor family (PAQR7, 8 and 5, respectively). The aim of this study was to determine the mRNA and protein expression levels of mPRα, mPRß and mPRγ through real-time PCR and Western blot analyses, respectively, and to determine the cellular localization of these proteins in the bovine endometrium and myometrium on days 2-5, 6-10, 11-16 and 17-20 of the oestrous cycle and weeks 3-5, 6-8 and 9-12 of pregnancy (n = 5/each time period). The resulting data showed the highest (P < 0.05) mPRα and mPRß mRNA expression in the endometrium on days 11-16 of the oestrous cycle compared to the other stages. In the myometrium, the level of mPRα mRNA was the lowest (P < 0.05) on days 6-16 of the oestrous cycle, while mPRß was the lowest on days 11-16. There were no changes (P > 0.05) in mPRγ mRNA expression in the endometrium and myometrium during the oestrous cycle. During pregnancy, in the endometrium and myometrium, the levels of mPRα and mPRß mRNA were comparable with those observed during the oestrous cycle. However, mPRγ mRNA expression was the highest (P < 0.001) during all stages of pregnancy compared with that observed during the oestrous cycle in both uterine tissues. The mPRα protein level only changed in the myometrium and was the highest (P < 0.05) during weeks 9-12 of pregnancy. However, in the endometrium, the expression of mPRß protein was higher (P < 0.05) on days 6-10 of the oestrous cycle than during weeks 6-8 of pregnancy. Strong positive immunoreactions for all mPR proteins were observed in the luminal and glandular epithelium but were less evident in the stromal cells and myocytes. In addition, all proteins were also localized in the endothelial cells of blood vessels in the uterus, suggesting that P4 may affect blood flow in this organ through mPRs. The presence of mPR receptors in the uterus indicates their participation in the regulation of uterine functions.

Methylation of progesterone receptor isoform A and B promoters in the reproductive system of cows.

The aim of this study was to investigate whether the promoters of progesterone receptor isoform A (PGRA) and B (PGRB) are methylated and to determine the percentage of methylation occurring for each isoform. Genomic DNA was isolated from the corpora lutea (CL) and endometrial slices from cows on Days 2-5, 6-10, 11-16 and 17-20 of the oestrous cycle. DNA was bisulphite-converted and amplified using methyl-specific polymerase chain reaction (PCR) with primers that detect both methylated and unmethylated sequences. The determination of the percentage of the methylation was performed using HpaII and MspI restriction enzymes. Methyl-specific PCR showed partial methylation of PGRA and PGRB promoters in the CL and endometrium during the oestrous cycle. Methylation for PGRA was between 15 and 17% and for PGRB was in the range of 6 to 7.7% during the oestrous cycle in the CL. In the endometrium, the methylation for PGRA was between 6 and 7.3% and for PGRB was between 3 and 4.8% during the oestrous cycle. The data obtained indicate that the higher promoter methylation of the PGRA isoform could be a mechanism for regulation of PGRA inhibitory activity against PGRB and, in this way, methylation may influence the regulation of progesterone action in the CL and endometrium.

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.

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.

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.

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.

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.

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 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.

Identification of optimal housekeeping genes for examination of gene expression in bovine corpus luteum.

The selection of proper housekeeping genes for studies requiring genes expression normalization is an important step in the appropriate interpretation of results. The expression of housekeeping genes is regulated by many factors including age, gender, type of tissue or disease. The aim of the study was to identify optimal housekeeping genes in the corpus luteum obtained from cyclic or pregnant cows. The mRNA expression of thirteen housekeeping genes: C2orf29, SUZ12, TBP, TUBB2B, ZNF131, HPRT1, 18s RNA, GAPDH, SF3A1, SDHA, MRPL12, B2M and ACTB was measured by Real-time PCR. Range of cycle threshold (C(t)) values of the tested genes varied between 12 and 30 cycles, and 18s RNA had the highest coefficient of variation, whereas C2orf29 had the smallest coefficient. GeNorm software demonstrated C2orf29 and TBP as the most stable and 18s RNA and B2M as the most unstable housekeeping genes. Using the proposed cut-off value (0.15), no more than two of the best GeNorm housekeeping genes are proposed to be used in studies requiring gene expression normalization. NormFinder software demonstrated C2orf29 and SUZ12 as the best and 18s RNA and B2M as the worst housekeeping genes. The study indicates that selection of housekeeping genes may essentially affect the quality of the gene expression results.

The role of the orphan receptor SF-1 in the development and function of the ovary.

The development of oocyte and ovulation require a precise synchronization at systemic and local levels. Nuclear receptors are involved in the regulation of these processes. In addition to the well-known nuclear receptors (e.g. receptors for estradiol, progesterone, glucocorticoids), a group of "orphan receptors" are distinguished within a receptor family. The orphan receptors are characterized by a lack of defined physiological ligands. Steroidogenic Factor 1 (SF-1, NR5A1) is a member of the orphan receptor group and is involved in the regulation of reproductive processes. The SF-1 structure is similar to that of the steroid receptors but does not have a modulatory domain. The SF-1 as a transcription factor may interact with genes in three main ways: a/ by a mechanism typical for nuclear receptors, encompassing homodimerization of SF-1 units, b/ by a formation heterodimers with other nuclear receptors, and c/ by action as a monomer. During fetal development, the SF-1, is responsible for differentiation of the gonads and, during the postnatal period, it is responsible for the increase in the expression of genes involved in steroidogenesis. Knock-out of SF-1 gene leads to a rapid death of newly born mice with symptoms of severe adrenal insufficiency. In humans, SF-1 dysfunction causes an adrenal insufficiency and infertility. Learning of the SF-1 and other orphan receptors' action mechanisms, will allow the creation of specific drugs, helpful in preventing some diseases of the female reproductive tract.

Involvement of prostaglandin F2alpha in the adverse effect of PCB 77 on the force of contractions of bovine myometrium.

Polychlorinated biphenyls (PCBs) stimulate in vitro both the force of myometrial contractions and endometrial secretion of PGF2alpha in cattle. Therefore, the goal of this study was to investigate the participation of PGF2alpha in the effect of PCBs on uterine contractility. For this aim, the myometrial strips were incubated (48h) with PCB 77 at the dose of 1, 10 and 100ng/ml (i.e., 0.0034, 0.034 and 0.34nmol/ml) separately or jointly with indomethacin (INDO, 10(-4)M), which blocks the PGF2alpha synthesis. Next, the force of myometrial strips contractions was measured. Further, the influence of PCB 77 (0.1, 1 and 10ng/ml) on the PGF2alpha secretion from myometrial cells after 6, 24, and 48h and PCB 77 (1 and 10ng/ml) on the mRNA expression of cyclooxygenase 2 (COX-2) and PGF2alpha synthase (PGFS) in myometrial cells after 6 and 24h, was investigated. The increase (P<0.05-0.001) of the contractions force of myometrial strips evoked by each dose of PCB 77, was markedly reduced (P<0.05-001) by INDO. There was an increase (P<0.05-0.001) of both PGF2alpha secretion after all studied periods of cell incubation and mRNA expression for COX-2 and PGFS after 6h treatment of myometrial cells with PCB 77. It can be concluded that myometrial synthesis of PGF2alpha and its further secretion is a part of the mechanism by means of which PCB 77 may affect the force of myometrial contractions in cattle.