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Expression of Progesterone Receptor Membrane Component 1, Serpine Mrna Binding Protein 1 and Nuclear Progesterone Receptor Isoforms a and B in the Bovine Myometrium During The

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Expression of Progesterone Receptor Membrane Component 1, Serpine Mrna Binding Protein 1 and Nuclear Progesterone Receptor Isoforms a and B in the Bovine Myometrium During The Journal of Reproduction and Development, Vol. 58, No 3, 2012 —Original Article— Expression of Progesterone Receptor Membrane Component 1, Serpine mRNA Binding Protein 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 proteins, 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 genes, 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 gene 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.
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