Robert Rekawiecki

Direct Inhibitory Effect of Progesterone on Oxytocin-Induced Secretion of Prostaglandin F2α from Bovine Endometrial Tissue

Abstract The effect of progesterone on oxytocin-induced secretion of prostaglandin (PG) F2α from bovine endometrial tissue explants was examined. Endometrial tissue from the late luteal phase were preincubated for 20 h in control medium. Explants were then treated for 6 h with control medium, oxytocin (10−7 M), progesterone (10−5 M), or both hormones. Oxytocin increased the medium concentration of 13,14-dihydro-15-keto-PGF2α, whereas progesterone completely suppressed the stimulatory effect of oxytocin. In experiment 2, isolated endometrial epithelial cells were incubated with progesterone (10−5 M), oxytocin (10−7 M), and combinations of these hormones with or without actinomycin D (1 ng/ml). Only oxytocin stimulated secretion of PGF2α, and this response was suppressed by progesterone. Oxytocin induced a rapid increase in intracellular concentrations of Ca2+ detected within 1 min of exposure of epithelial cells from the same cows. Progesterone pretreatment diminished this response. In experiment 3, direct effects of progesterone (2 nM–20 μM) on binding of 3H-oxytocin to the membrane preparation from epithelial cells were determined by saturation analysis. Oxytocin binding was suppressed by progesterone at every dosage tested. Progesterone is capable of suppressing the ability of oxytocin to induce endometrial secretion of PGF2α. This effect appears to be mediated through a direct interference in the interaction of oxytocin with its own receptor.

Neural regulation of the bovine corpus luteum.

The ovarian noradrenergic stimulation or noradrenaline (NA) administration directly to the ovary in cow increases ovarian oxytocin (OT) release and post-translational processing of OT synthesis within a few minutes has been established in both in vivo and in vitro studies. Furthermore, NA affects progesterone secretion and its synthesis by an increase of cytochrome P450scc and 3beta-hydroxysteroid dehydrogenase activity. This effect is mediated via luteal cell beta(1)- and beta(2)-receptors. Their total amount correlates with peripheral progesterone concentrations during the luteal phase and this reflects the ability of the ovary to react to beta-stimulation. On the other hand, ovarian denervation causes a decrease of steroidogenic activity in the CL, an increase of beta-receptors on luteal cells, a delay in follicular development and the disruption of cyclicity. Moreover, decrease of progesterone secretion by 20-30% was seen after brief pharmacological blockade of ovarian beta-receptors in the mid-cycle of cattle. We assume that tonic beta-stimulation of the CL ensures the basal secretion of progesterone, whereas acute noradrenergic activation supports the CL during stressful situations which could impair its function. Conversely, long-lasting increase in blood catecholamine concentrations markedly decreases the number of beta-receptors in CL, presumably due to their down-regulation. Concentrations of dopamine (DA) within the CL are highly correlated with those of NA during the estrous cycle, and are higher in the newly-formed than in the developed corpus luteum, the regressed corpus luteum or the corpus luteum of pregnant females. Bovine CL can synthesise de novo NA from DA as a precursor. Concluding, presented data indicate that noradrenergic stimulation can be an important part of mechanism supporting secretory function of CL.

Role of prostaglandin E2 in basal and noradrenaline-induced progesterone secretion by the bovine corpus luteum.

The role of prostaglandin E2 (PGE2) in basal and noradrenaline (NA)-stimulated utilization of high density lipoprotein (HDL) as a source of cholesterol for progesterone synthesis was examined. In Experiment 1, a cannula was inserted into the aorta abdominalis through the coccygeal artery (cranial to the origin of the ovarian artery) in mature heifers, to facilitate infusion of NA (4 mg/30 min; n = 3) on day 10 of the estrous cycle. Three other heifers were similarly cannulated to serve as control. Before, during, and after NA or saline infusion, blood samples from the vena cava were collected every 5-15 min for analysis of PGE2, progesterone, and cholesterol. Each NA infusion stimulated (P < 0.01) secretion of both hormones in heifers. Short-duration increases (P < 0.05) in progesterone were observed due to the infusion of NA while cholesterol was not altered significantly. In addition, increases in PGE2 concentrations (P < 0.05) compared to controls were seen after NA infusion. Therefore, we used an in vitro model to verify the effect of PGE2 on HDL utilization by luteal cells from day 5 to 10 of the estrous cycle. In the preliminary experiment, 10(-6) M of PGE2 out of four different doses examined was selected for further studies, since it evoked the highest release of progesterone. In the next experiment, it was found that HDL increases progesterone secretion by luteal cells and both PGE2 and LH increased (P < 0.05) the response to HDL while NA did not. In the last in vitro experiment, progesterone stimulated PGE2 secretion by luteal cells. In conclusion, PGE2 may be directly involved in the utilization of cholesterol from HDL for progesterone synthesis. Furthermore, PGE2 may influence NA-stimulated progesterone secretion by the corpus luteum (CL). It is concluded that there is a positive feedback loop between progesterone and luteal PGE2 during days 5-10 of the estrous cycle.

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.

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.

Involvement of prostaglandin F2α 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.

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.

The expression of Steroidogenic Factor-1 and its role in bovine steroidogenic ovarian cells during the estrus cycle and first trimester of pregnancy

The orphan receptor Steroidogenic Factor-1 (SF-1, NR5A1), a member of the nuclear receptor superfamily, is present in fetal and adult steroidogenic tissues and also participates in the regulation of ovarian function. In this study, the expression levels of SF-1 mRNA and protein were determined in granulosa cells (from follicles >1cm and <1cm in diameter) and luteal tissue (from days 1-5, 6-10, 11-15, and 16-19 of the estrous cycle and weeks 3-5, 6-8, and 9-12 of pregnancy). Additionally, the effects of a synthetic SF-1 stimulator (4-(heptyloxy)phenol - HxP; 1×10(-7)M) and a synthetic SF-1 inhibitor (F0160; 1×10(-5)M) on the secretion of estradiol and oxytocin (OT) from granulosa cells (from follicles>1cm) and the secretion of progesterone (P4) and OT from luteal cells (days 11-16 of the estrous cycle) were investigated. The levels of SF-1 mRNA and protein were higher in granulosa cells (P<0.05) from follicles>1cm than in cells from follicles<1cm. In luteal tissue, the mRNA abundance was the highest (P<0.05) on days 6-10 of the estrous cycle, and the amount of protein was the highest on days 6-15 (P<0.05). The lowest levels of mRNA and protein for SF-1 were observed on days 16-19 of the estrous cycle (P<0.05). The abundance of SF-1 mRNA decreased at 9-12 weeks of pregnancy (P<0.05). The stimulation of the studied cells with HxP increased P4 and estradiol secretion from luteal and granulosa cells, respectively, and OT secretion from both types of cells. The SF-1 inhibitor did not affect hormone secretion by either type of cell, but it did diminish the effect induced by the SF-1 stimulator. The obtained data revealed estrous cycle-dependent levels of mRNA and protein for SF-1 in luteal tissue, and the use of a specific SF-1 stimulator and a specific SF-1 inhibitor confirmed the involvement of this receptor in steroidogenesis and OT secretion from cultured granulosa and luteal cells. These findings suggest that the SF-1 receptor participates in the local regulation of ovarian function during both the estrous cycle and the first trimester of pregnancy in cows. Furthermore, the concentrations of the SF-1 inhibitor and stimulator that we used in the primary cell culture could effectively modify the activity of this receptor.

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.