Journal of Reproduction and Development, Vol. 58, No 3, 2012 —Original Article— Expression of Progesterone Receptor Membrane Component 1, Serpine mRNA Binding 1 and Nuclear Progesterone Receptor Isoforms A and B in the Bovine Myometrium During the Estrous Cycle and Early Pregnancy Dominika Slonina1), Magdalena K. Kowalik1) and Jan Kotwica1) 1)Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, 10-747 Olsztyn, Poland 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. Key words: Cow, Myometrium, Nongenomic effect, Progesterone, Progesterone receptor membrane component 1 (PGRMC1), Serpine mRNA binding protein 1 (SERBP1) (J. Reprod. Dev. 58: 288–294, 2012)

rogesterone (P4), which is produced in the corpus luteum is not fully understood. It has been suggested that P4, a lipophilic P(CL), regulates various female reproductive functions. This substance, can modify the fluidity of the cell membrane and thus hormone is also responsible for morphological, functional and change the affinity of membrane receptors for their ligands [9, 13, structural changes in the endometrium during the luteal phase 14]. Moreover, P4 can be bound by its specific membrane receptor and for suppression of myometrial contractions, which prepares to stimulate early intracellular signalling pathways and initiate a the uterus for blastocyst implantation and ensures maintenance of specific cellular response [5, 8]. There are several putative membrane pregnancy [1]. Many functions of P4 are mediated through binding P4-binding , including: nPRs, a novel family of membrane to its specific nuclear progesterone receptor (nPR), which acts as a progestin receptors (mPRs), and progesterone receptor membrane ligand-transcription factor and is expressed in two main isoforms, component 1 (PGRMC1), which may form an active complex with A (PRA) and B (PRB) [2]. PRA (94 kDa) is about 164 amino acids serpine mRNA binding protein 1 (SERBP1) [5–8]. shorter than PRB (120 kDa) in humans [2], and a similar molecular It has been suggested that the nongenomic effect of P4 observed mass was found in cattle [3, 4]. The different isoforms of nPR play in the uterus is mediated via the 28 kDa PGRMC1 protein [5, 6], different roles in the cells. Isoform B acts mainly as an activator which contains a short transmembrane domain and a cytochrome of progesterone-responsive , while PRA can inhibit the b5 binding domain, and is structurally different from both nPRs activity of PRB and other nuclear receptors such as the estrogen, and mPRs [5, 15]. It has been found that PGRMC1 protein is in- glucocorticoid and mineralocorticoid receptors [2]. volved in steroidogenesis and cellular homeostasis [16], and also in P4 can also exert its effects more directly, by rapid, nongenomic reproductive functions like the anti-apoptotic effects of P4 in the pathways [5–7]. This nongenomic effect of P4 has been found in ovary [17–19], or its effect on contractility of the myometrium [20]. a number of tissues from the female reproductive tract [5, 7, 8] in PGRMC1 can bind to the SERBP1 protein (50 kDa) and form a P4 mammals, including cows [9–12], but the nature of this mechanism receptor-membrane complex, and P4 can evoke its anti-apoptotic and mitotic effect on cells [8, 17]. Activation of this complex by P4 causes an increase in cAMP levels and activation of protein Received: April 13, 2011 kinase G, which leads to a reduction in Ca2+ levels in cells [8, 19]. Accepted: December 22, 2011 We have shown that P4 decreased intracellular Ca2+ mobiliza- Published online in J-STAGE: January 25, 2012 ©2012 by the Society for Reproduction and Development tion in bovine myometrial cells [21] and that P4 inhibited oxytocin Correspondence: J Kotwica (e-mail: [email protected]) (OT)-stimulated PGF2α and PGE2 secretion from these cells by EFFECT OF PROGESTERONE ON THE BOVINE MYOMETRIUM 289 both genomic and nongenomic pathways [12]. So, it can be assumed total reaction volume of 20 µl containing 50 mM Tris–HCl (pH that the latter effect of P4 was evoked partly via a membrane pro- 8.3), 75 mM KCl, 3 mM MgCl2, 5 mM dithiothreitol, 10 mM dNTP gesterone receptor, PGRMC1, found in the human [20] and mouse mix, 1 µg oligo(dT)23 primers (Fermentas, Vilnius, Lithuania), myometrium [22]. Since P4 is essentially involved in the control of and 200 IU of M-MLV reverse transcriptase (Promega, Madison, myometrial growth and its contractility, the aim of the present study WI, USA). RNA was denatured at 70 C for 10 min, and then the was (1) investigate the expression of PGRMC1, SERBP1 and PR RT reaction was carried out at 42 C for 60 min. The reaction was mRNA; and (2) to evaluate the protein levels of PGRMC1, SERBP1 terminated by heating for 10 min at 70 C. The cDNA was stored and PR isoforms A and B in the bovine myometrium during the (–20 C) until real-time PCR amplification. estrous cycle and the first trimester of pregnancy. Real-time PCR quantification Material and Methods Real-time PCR was performed with an ABI Prism 7300 se- quence detection system using Power SYBR Green PCR master Tissue collection mix (Applied Biosystems, Foster City, CA, USA). Based on Uteri (ipsilateral to the ovary with a CL) were collected from sequences in GenBank (NCBI), primers for PGRMC1 and SERBP1 cows at a commercial slaughterhouse on days 1–5, 6–10, 11–16, and were designed using the Primer Express 3.0 software (Applied 17–21 (n=6 for each stage) of the estrous cycle and during weeks Biosystems). Primers for PR and GAPDH (reference gene) were 3–5, 6–8, and 9–12 (n=5 per stage) of pregnancy within 20 min of previously published [26]. All primers were synthesized by IBB killing the animals. Days of the estrous cycle were estimated by PAN (Warsaw, Poland). Primer sequences, expected PCR products morphological observations of the ovaries and uterus as previously length and GenBank accession numbers of references are listed in described [23], while the stages of pregnancy were estimated as Table 1. Each PCR reaction well (final volume of 25 μl) contained previously published by Jainudeen and Hafez [24]. All materials 5μl of diluted cDNA (200 ng), 200 pM each of forward and reverse used in these studies were obtained from Sigma-Aldrich Chemical primers and 12.5 μl SYBR Green PCR master mix. Serial dilutions (St. Louis, MO, USA) unless otherwise stated. of the appropriate cDNA were used as standard curves for gene quantification. Real-time PCR was carried out as follows: initial Expression of PGRMC1, SERBP1 and PR mRNA in the denaturation (10 min at 95 C) followed by 40 cycles of denaturation bovine myometrium (15 sec at 95 C) and annealing and extension (60 sec at 60 C). All Immediately after uteri collection, myometrial tissue was sepa- reactions were performed in duplicate. After each PCR reaction, rated from the endometrium, snap frozen in liquid nitrogen and melting curves were obtained by stepwise increases in temperature stored at –80 C until further use. The deeply frozen tissues were from 60 C to 95 C to ensure single product amplification. The speci- homogenized with a vibratory mill (Retsch MM-2). The tissue ficity of the product was also confirmed by gel electrophoresis and powder was divided into individual portions for isolation of RNA sequencing. Data obtained from the real-time PCR for PGRMC1, and subsequent cDNA synthesis. SERBP1 and PR were normalized to the GAPDH mRNA content.

Expression of PGRMC1, SERBP1, PRA and PRB proteins Western blot analysis and the P4 concentration in the bovine myometrium Total protein (100 µg) was dissolved in SDS gel-loading buf- Myometrial tissue samples (500 mg) from defined days/weeks fer (50 mM Tris-HCl, pH 6.8; 4% SDS; 20% glycerol; and 2% of the estrous cycle or pregnancy were homogenized in ice-cold β-mercaptoethanol), heated to 95 C for 8 min and separated on homogenization RIPA buffer (25 mM Tris-HCl, pH 7.6, 150 mM 12% (for PGRMC1) and 10% (for SERBP1 and PR isoforms) NaCl, 1% Triton X-100, 1% sodium deoxycholate, 5 mM EDTA SDS-PAGE gels. Separated proteins were transferred onto a 0.2 and 0.1% SDS) containing 12 µl/ml Protease Inhibitor Cocktail μm nitrocellulose membrane in transfer buffer (20 mM Tris-HCl, and further homogenized on ice with an ultrasound homogenizer pH 8.2; 150 mM glycine; and 20% methanol). Nonspecific binding (Sonics & Materials, Newton, CT, USA). Homogenates were then sites were blocked with 5% nonfat dry milk in TBS buffer (50 mM centrifuged for 10 min at 1000× g at 4 C, and the supernatant, Tris–HCl; 0.9% NaCl) containing 0.05% Tween-20 for 1.5 h at room which contained the cytosol and membrane fractions, was col- temperature. The membrane was then incubated overnight at 4 C lected, aliquoted and stored at –80 C for Western blot analysis. with the following primary antibodies: rabbit anti-PGRMC1 at a The protein concentration was determined by the Bradford [25] dilution of 1:250 (gift from Prof M Wehling, Clinical Pharmacology, method. The progesterone concentration after extraction from University of Heidelberg, Mannheim, Germany), monoclonal tissue was determined by enzyme immunoassay (EIA). mouse anti-SERBP1 at a dilution of 1:500 (Abcam, Cambridge, UK) and polyclonal rabbit anti-PR at a dilution of 1:200 (Santa Total RNA isolation and cDNA synthesis Cruz Biotechnology, Santa Cruz, CA, USA). The membrane was Total RNA was extracted from myometrial tissue using a Total then incubated with secondary goat anti-rabbit (dilution 1:30,000) RNA Prep Plus Kit (A&A Biotechnology, Gdansk, Poland) accord- or anti-mouse (dilution 1:30,000) antibody conjugated to alkaline ing to the manufacturer’s instructions. The RNA concentration and phosphatase. Between each step, membranes were washed in TBST quality were determined using a NanoDrop 1000 spectrophotometer buffer. Immunoreactive bands were detected using a visualization (Thermo Scientific, Wilmington, DE, USA). Total RNA (1 µg) was stock solution (NBT/BCIP with alkaline phosphatase assay buf- treated with DNase and reverse transcribed (RT) into cDNA in a fer containing 0.1 M NaCl, 5 mM MgCl2, and 100 mM Tris–HCl, 290 SLONINA et al.

Table 1. Forward and reverse primer sequences, amplicon sizes in base pairs and GenBank accession numbers/ references for real-time PCR reactions

pH 9.5). Western blots were quantitated using the Kodak EDAS 290 Software (Eastman Kodak, Rochester, NY, USA). To verify equal protein loading, membranes were reprobed with monoclonal mouse anti-GAPDH antibody (concentration 0.1 µg/ml). In our experiment, the anti-PGRMC1 antibody detected two bands at approximately 25 kDa and 23 kDa in both the cyclic and pregnant myometrium (Fig. 2a). This was in agreement with the findings described by Zhang et al. [22], who used the same antibody. The SERBP1 antibodies recognized one band of ~50 kDa in lysates derived from the myometrial samples (Fig. 2b). To test the specific- ity of the antibody for SERBP1, we used the positive control for SERBP1 antibody (HeLa whole cell lysate; ab29545, Abcam). The PR antibodies detected multiple bands of the nuclear P4 receptor using the blocked peptide for PR antibody (sc-538P; Santa Cruz Biotechnology) to allow for identification of the PR-B (116 kDa) and PR-A (81 kD) receptors (Fig. 3a, b).

Determination of P4 concentration Progesterone was extracted from myometrial tissues with pe- troleum ether (efficiency above 85%), and the concentration was determined by EIA [27] using horseradish peroxidase-labelled P4 as a tracer and P4 antiserum as previously characterized [28]. The range of the standard curve was 0.1– 25 ng/ml, and the sensitivity of the procedure was 0.15 ng/ml. The intra- and interassay coef- ficients of variation were 8.7% and 11%, respectively.

Statistical analysis All data were expressed as the mean ± SEM. Statistical analyses were conducted using one-way ANOVA for repeated measures and followed by a Newman-Keuls post hoc test (GraphPad PRISM 4.0; Graph Pad Software, San Diego, CA, USA) after testing for normality. Pearson’s coefficients of correlation (r) between mRNA and proteins expression and P4 concentrations were calculated.

Results

Fig. 1. Mean (± SEM) expressions of (a) PGRMC1 (n=5–6), (b) Expression of PGRMC1, SERBP1 and PR mRNA in the SERBP1 (n=5–6) and (c) PR (n=5–6) mRNA as determined bovine myometrium by real-time PCR in the bovine myometrium during the The expression of PGRMC1 and SERBP1 mRNA in the bovine estrous cycle and pregnancy. All values are expressed as a myometrium during all stages of the estrous cycle and early ratio of receptor expression to GAPDH. Bars with different superscripts are significantly different (P<0.05). EFFECT OF PROGESTERONE ON THE BOVINE MYOMETRIUM 291

Fig. 3. Mean (± SEM) expressions of PR (a) isoform B (n=5–6) and (b) isoform A (n=5–6) proteins as determined by Western blot analysis in the bovine myometrium during the estrous cycle and pregnancy. All values are expressed as a ratio of receptor Fig. 2. Mean (± SEM) expressions of (a) PGRMC1 (n=5–6) and expression to GAPDH protein. Upper panels are representative (b) SERBP1 (n=5–6) proteins as determined by Western blots of PR and GAPDH antibodies. Lower panels are blot analysis in the bovine myometrium during the estrous densitometric analyses of PRB and PRA. Bars with different cycle and pregnancy. All values are expressed as a ratio of superscripts are significantly different (P<0.05). receptor expression to GAPDH protein. Upper panels are representative blots of PGRMC1, SERBP1 and GAPDH antibodies. Lower panels are densitometric analyses of PGRMC1 and SERBP1. Bars with different superscripts are levels for PGRMC1 were found in pregnant animals (3–5 weeks), significantly different (P<0.05). and the lowest levels of this protein were found during weeks 9–12 of pregnancy and after day 11 of the estrous cycle (Fig. 2a). There were no significant differences (P>0.05) in myometrial SERBP1 pregnancy (Fig. 1a, b) was determined. There were no observed protein expression during the estrous cycle and early pregnancy changes (P>0.05) in the PGRMC1 mRNA level during the estrous (Fig. 2b). PRA and PRB had similar profiles of protein expression cycle (Fig. 1a), while the mRNA expression of SERBP1 and PR was in the bovine myometrium (Fig. 3). The highest (P<0.05) protein lower (P<0.05) on days 11–16 of estrous cycle than on other days of levels of both isoforms of the nuclear P4 receptor were observed the cycle (Fig. 2b, c). Higher (P<0.05) mRNA levels of PGRMC1, on days 1–5 of the estrous cycle (Fig. 3a, b). After day 5 of the SERBP1 and PR were found during stages of pregnancy relative estrous cycle, the expression of PRA and PRB proteins decreased to the days of estrous cycle (Fig. 1). (P<0.05) to levels similar to those measured during early preg- nancy (Fig. 3a, b). Expression of PGRMC1, SERBP1, PRA, and PRB proteins in the bovine myometrium P4 concentration Immunoblotting of myometrial homogenates showed a major Myometrial P4 concentrations during different days of the es- band at approximately 25 kDa for PGRMC1 (Fig. 2a), 50 kDa for trous cycle and the first trimester of pregnancy are shown in Fig. SERBP1 (Fig. 2b) and 116 kDa and 81 kDa for the two isoforms of 4. Progesterone levels were highest on days 1–5 (P<0.05) of the nuclear P4 receptor, PRB (Fig. 3a) and PRA (Fig. 3b), respectively. estrous cycle. After day 6 of the estrous cycle, the concentration of The levels of the PGRMC1 protein did not change (P>0.05) during this hormone in the tissue decreased (P<0.05) and was maintained the estrous cycle (Fig. 2a). The highest (P<0.05) protein expression at a similar level during the pregnancy (Fig. 4). Additionally, the 292 SLONINA et al.

maintenance of pregnancy. This study has shown variable expression levels of mRNA for SERBP1 in the myometrium during the estrous cycle and pregnancy, while the level of SERBP1 protein was not changed. Higher mRNA expression for SERBP1 and PGRMC1 was found during gestation relative to the estrous cycle. Therefore, it is possible that a PGRMC1/ SERBP1 complex is involved in nongenomic P4 action and can play a role in the maintenance of pregnancy in cows. It has been suggested that P4 impairs binding of OT to its membrane receptor via a nongenomic mechanism [9, 13, 14] and thus diminishes levels of intracellular Ca2+ and decreases prostaglandin secretion from the bovine endometrium and myometrium [10, 12]. However, the Fig. 4. Mean (± SEM) progesterone (P4) concentration nongenomic effect of P4 on the OT receptor was not confirmed in bovine myometrium collected from different by other authors [31, 32]. Thus, P4 may be bound by a complex of stages of the estrous cycle and pregnancy (n=5–6). PGRMC1 and SERBP1 proteins, which causes an increase in the Values with different superscripts are significantly cGMP level and subsequent activation of protein kinase G. This different (P<0.05). leads to the reduction of calcium mobilization [8, 19] and thus may reduce PGF2α secretion from the cells [10, 11]. Furthermore, the nongenomic action of P4 could block calcium ion channels and concentration of P4 was positively correlated (P<0.001) with the prevent smooth muscle contraction in both the mouse [33, 34] and expression of the PRA and PRB proteins during the estrous cycle human [20] myometrium. Therefore, it is possible that the binding (r=0.70 and r=0.74) and pregnancy (r=0.79 and r=0.86), respectively. of P4 to the PGRMC1/SERBP1 protein complex decreases myome- trial contractions and may take part in many processes promoting Discussion the development and maintenance of pregnancy such as cell dif- ferentiation, regulation of cell apoptosis and steroidogenesis in the The results of this study demonstrate the expression of mRNA uterus [20, 22]. Moreover, our earlier studies [35] confirmed the and protein for putative P4 membrane protein receptor PGRMC1 presence of the PGRMC1 and SERBP1 proteins in myocytes and in and its binding partner SERBP1, as well as nuclear PRs in the the endothelium of blood vessels in the bovine myometrium during bovine myometrium during the estrous cycle and the first trimes- the estrous cycle and the first trimester of pregnancy. This suggests ter of pregnancy. The PGRMC1 mRNA level in the myometrium that P4 may modulate blood flow in the female reproductive tract was relatively constant during the estrous cycle, but the levels by a direct effect on blood vessels via a nongenomic mechanism. significantly increased during the first trimester of pregnancy. It should be noted that PGRMC1 and SERBP1 proteins were also Myometrial PGRMC1 protein expression levels also depend on the found in vascular smooth muscle cells in the human placenta [22]. stage of the estrous cycle and gestation. Thus, it may be assumed Hence, it is suggested that a complex of these proteins could be that P4 acts on the cells directly by binding to PGRMC1 protein involved in the nongenomic effect of P4 on the uterus and in this as previously suggested [22], but the intensity of this process may way take part in the maintenance of pregnancy. depend on the female’s physiological status. In this study, expres- The highest levels of mRNA expression for PR in the myome- sion of the PGRMC1 protein was relatively high at the beginning trium were found during all stages of gestation, while the lowest of the estrous cycle, and this may be related to the high P4 con- expression levels of this gene were observed on days 11–16 of the centration in the myometrium on days 1–5 of the estrous cycle. estrous cycle. It is possible that this reduction in PR mRNA expres- Indeed, it was found that P4 increased PGRMC1 mRNA levels in sion during the luteal phase is correlated with the high levels of ovariectomized mice, which may promote the differentiation of P4 in peripheral blood in cows, as previously suggested [36, 37]. uterine cells [22]. We also found that the level of PGRMC1 protein It was found that genomic activity of P4 inhibited PGF2α secre- increased during pregnancy, compared with the estrous cycle. tion by diminishing the concentration of OT receptors [38, 39]. This suggests that this receptor may be crucial for maintenance It has been suggested that a high concentration of P4 for 10–12 of pregnancy. This suggestion was confirmed by Wu et al. [20], days in cows is essential for abolition of the inhibitory effect of who showed that PGRMC1 activation may inhibit contractility of P4 on OT receptor expression and for the subsequent increase in the myometrium during pregnancy and that the downregulation endometrial susceptibility to OT action [40]. This may initiate of PGRMC1 expression is associated with term and preterm labor the first pulse of PGF2α and the subsequent cascade reaction that in humans. However, other studies [29] have shown that changes switches on luteolysis [41]. High mRNA expression for PR during in the membrane progestin receptors could also contribute to the early pregnancy may be positively correlated with the secretion of functional P4 withdrawal in the human myometrium during labor, specific substances by the embryo, as found in the endometrium but the role of these receptors in transducing P4 signals has also of ruminants [42, 43]. Whether this mechanism operates in the been questioned [30]. Therefore, we suggest that P4 by interaction myometrium needs to be elucidated. with PGRMC1 may affect growth, differentiation and contractility The highest protein levels for PR isoforms A and B were observed of myometrial cells and in this way promote the development and on days 1–5 of the estrous cycle. Protein levels for both isoforms EFFECT OF PROGESTERONE ON THE BOVINE MYOMETRIUM 293

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