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Mol. Hum. Reprod. Advance Access originally published online on October 31, 2007
Molecular Human Reproduction 2007 13(12):845-851; doi:10.1093/molehr/gam075
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© The Author 2007. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Hormonal regulation of G{alpha}i2 and mPR{alpha} in immortalized human oviductal cell line OE-E6/E7

Kati S. Mönkkönen1,2, Reza Aflatoonian2, Kai-Fai Lee3, William S.B. Yeung3, Sai-Wah Tsao4, Jarmo T. Laitinen5 and Alireza Fazeli2,6

1Department of Pharmacology and Toxicology, University of Kuopio, FIN-70211 Kuopio, Finland 2Academic Unit of Reproductive and Developmental Medicine, The University of Sheffield, Level 4, Jessop Wing, Tree Root Walk, Sheffield S10 2SF, UK 3Department of Obstetrics and Gynaecology, The University of Hong Kong, Pokfulam, Hong Kong 4Department of Anatomy, The University of Hong Kong, Pokfulam, Hong Kong 5Institute of Biomedicine, Department of Physiology, University of Kuopio, FIN-70211 Kuopio, Finland

6 Correspondence address. Tel: +44-114-226-8195; Fax: +44-114-226-8538; E-mail: a.fazeli{at}sheffield.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Funding
 Acknowledgements
 References
 
Heterotrimeric G proteins play a key role in membrane-mediated cell-signalling and hormonal regulation. Our earlier studies gave evidence of G protein subunit G{alpha}i2 being under hormonal regulation in human in vivo. In this study, we used immortalized human oviduct epithelial cell line OE-E6/E7 as a model to study the hormonal regulation of G{alpha}i2. We aimed at clarifying whether estradiol or progesterone could individually regulate the expression of G{alpha}i2 and its potential signalling partners. Furthermore, we aimed to investigate which sex hormone receptors could potentially mediate the gene regulation in OE-E6/E7 cell line. OE-E6/E7 cells were cultured for 5 days with different concentrations of estradiol or progesterone. Quantitative real-time polymerase chain reaction (Q-PCR) was performed using cDNA of the hormone-treated cells to reveal any changes in gene expression. The presence of potential receptor targets in these cells was studied using PCR. Our data clearly showed that low concentrations of estradiol up-regulated the expression of G{alpha}i2 gene and down-regulated the expression of membrane progesterone receptor mPR{alpha} gene in OE-E6/E7 cell line. Progesterone had no significant effect on G{alpha}i2 gene expression, but it caused up-regulation of mPR{alpha} gene expression. In conclusion, it appears that sex hormones regulate the expression of G{alpha}i2 and mPR{alpha} genes in a reverse manner in OE-E6/E7 cells. Our results suggest that estrogen receptor ERβ mediates the regulatory effects of estradiol in these cells.

Key words: estradiol/fallopian tube/G protein/progesterone/regulation


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Funding
 Acknowledgements
 References
 
Fallopian tubes have a crucial role in early events of fertilization. They act as sperm reservoirs (Pacey et al., 1995; Baillie et al., 1997), enhancing motility and preserving viability of sperm (Murray and Smith, 1997). Furthermore, they play an important role in sperm capacitation (Kervancioglu et al., 1994, 2000). Epithelial cells on the luminal surface of Fallopian tubes are of special importance in fertility, as they are in direct cell-to-cell contact with gametes. Epithelial cells consist of two cell types, ciliated cells and non-ciliated secretory cells. Motility of the epithelial cilia is essential in gamete transport in association with tubal secretory flow and muscle contractility (Jansen, 1984). Epithelial secretory cells produce and secrete oviductins, oviduct-specific glycoproteins (Verhage et al., 1988; Wagh and Lippes, 1989; Arias et al., 1994), thus providing an optimal environment for fertilization and stimulating the early development of the embryo (Liu et al., 1995; Yeung et al., 1996).

Although the physiological roles of Fallopian tubes and the luminal epithelial cells are well established, knowledge about the signalling pathways involved remains limited. G protein-coupled receptors are the most widespread family of cell-surface receptors and their intracellular signalling partners, heterotrimeric G proteins, are recognized as crucial elements in various types of membrane-mediated cell-signalling. In reproductive endocrinology, G protein-coupled receptors have a regulatory role in gonadotrophin-releasing hormone (GnRH)-induced secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) from the anterior pituitary gland (Tsutsumi et al., 1992; Chi et al., 1993). In gonads, G protein-coupled receptors regulate the synthesis and secretion of sex hormones by mediating gonadotrophin signalling (Loosfelt et al., 1989; McFarland et al., 1989; Minegishi et al., 1990, 1991; Sprengel et al., 1990). Heterotrimeric G proteins are classified into four families (Gs, Gi, Gq and G12) (Hepler and Gilman, 1992). Gi family proteins are the most diverse and interact with a wide variety of G protein-coupled receptors, such as GnRH (Hawes et al., 1993; Stanislaus et al., 1998; Krsmanovic et al., 2001, 2003), FSH (Arey et al., 1997) and LH receptors (Herrlich et al., 1996). Recently, Gi family proteins have also been demonstrated to interact with cell-surface receptors for estrogen (Benten et al., 2001) and progesterone (Zhu et al., 2003b; Karteris et al., 2006), mediating the signal transduction of their rapid, non-genomic actions.

During the menstrual cycle, Fallopian tubes undergo cyclic changes. Ovarian steroid hormones estrogen and progesterone change the morphology of luminal epithelial cells, alter the intraluminal environment and promote tubal activity and functions (Verhage et al., 1979). Estrogen is responsible for epithelial cell differentiation, hypertrophy and secretory activity as well as ciliogenesis. Progesterone induces atrophy and deciliation (Verhage et al., 1979; Donnez et al., 1985). Largely due to limited access to Fallopian tube cells, the information on specific signalling components involved in their hormonal regulation is insufficient.

Our recent study showed specific localization of G protein subunit G{alpha}i2 in Fallopian tube epithelial cells and the cilia. Further, our data gave evidence of G{alpha}i2 being under hormonal regulation in female reproductive tract in vivo (Mönkkönen et al., 2007a). In the present study, we used immortalized human oviductal epithelial cell line OE-E6/E7 as a valuable model of Fallopian tube epithelium to study hormonal regulation of selected signalling proteins. We aimed at clarifying whether estradiol or progesterone could individually regulate the expression of G protein subunit G{alpha}i2 and its potential signalling partners such as membrane progesterone receptor mPR{alpha}. Additionally, we aimed at identifying the potential nuclear receptors mediating hormonal regulation in these cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Funding
 Acknowledgements
 References
 
Cell culture
Immortalized Human Fallopian tube cell line (OE-E6/E7) (Lee et al., 2001) was cultured at +37°C in DMEM (F12) culture media (Invitrogen, Paisley, UK) supplemented with 1% penicillin and streptomycin (Sigma-Aldrich, Poole, UK), 10% fetal calf serum (Invitrogen) and L-glutamine (Invitrogen) in 5% CO2 atmosphere.

In the case of hormone treatment studies, OE-E6/E7 cells were cultured in triplicates at +37°C in DMEM (F12) culture media without serum and phenol red (Invitrogen) and supplemented with 1% penicillin and streptomycin (Sigma-Aldrich). Water-soluble estradiol (Sigma-Aldrich) was used at 0.1, 1, 10 and 100 nM concentrations. Water-soluble progesterone (Sigma-Aldrich) was used at concentrations of 1, 10, 100 and 1000 nM. In both experiments, control media was supplemented with cyclodextrin (Sigma-Aldrich), at a concentration equivalent to that present in hormone media. The cells were split into hormone media and cultured until confluency (for 5 days).

RNA isolation and cDNA synthesis
For genomic studies, OE-E6/E7 cells were washed with Ca2+ and Mg2+ free phosphate buffered saline (PBS), harvested using trypsin-EDTA (Invitrogen) and pelleted by centrifugation at 300g for 5 min. One milliliter of TRIreagent (Sigma-Aldrich) was added onto pellet (5 x 106 cells). Endometrial and Fallopian tube tissues used for control purposes in the present study were removed from RNAlater and homogenized in 3 ml of TRIreagent (Sigma-Aldrich) using an Ultra-Turrax homogenizer for 2 min. Total RNA from the tissues and pelleted cells stored in TRIreagent was extracted following standard protocol supplied by the manufacturer. Total RNA was treated with DNase I (DNA-freeTM, Ambion) to remove genomic DNA contamination from the samples. First-strand cDNA synthesis was performed using oligo dT primers (Metabion, Martinsried, Germany) and reverse transcription by SuperScript II (200 U/µl, Invitrogen). Negative controls were prepared without the enzyme (non-reverse transcribed controls, RT controls).

Polymerase chain reaction
PCR was performed with the constructed cDNAs, Platinum Blue PCR Super Mix (Invitrogen), and primers from Metabion. The primer pairs, amplified sequences and annealing temperatures used are described in detail in Table I. The primer sequences without reference were newly designed for this study. The amplification was run for 40 cycles under the following conditions: 95° 30 s, annealing temperature for each primer 30 s, 72° 30 s. All experiments included RT controls as well as negative controls (no cDNA). cDNA from endometrial and Fallopian tube tissues (Mönkkönen et al., 2007a) were used as control. PCR products were separated on 1.2% agarose gel.


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  		Table I. Primer sequences used for the study.

 
Quantitative real-time PCR
Quantitative real-time PCR (Q-PCR) was performed in triplicates with the constructed cDNAs and the same primers that were used in PCR reactions (Table I). SYBR Green Jump Start Taq Readymix (S 4438, Sigma-Aldrich) master mix [containing 10 µl SYBR Green, 7 µl of water, 1 µl of each primer (20 pmol) and 1 µl of cDNA] was added to each well of PCR plate and amplification was performed under the following conditions: 50 cycles (95° 30 s, annealing temperature of each primer used 30 s, 72° 30 s). All experiments included RT controls and negative controls (no cDNA).

Results were analyzed using iCycler (Biorad laboratories Ltd, Hemel Hempstead, UK). The threshold cycle values were normalized against threshold value of human β-actin. The results were expressed as mean ± SEM. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparison test. P < 0.05 was considered as statistically significant difference.

Western blot analysis
Immortalized Fallopian tube epithelial cells were washed with Ca2+ and Mg2+ free PBS, harvested using trypsin-EDTA (Invitrogen) and pelleted by centrifugation at 1000g for 5 min. The resulting pellet was snap frozen and stored –70°C. Pelleted cells were thawed by addition of ice-cold sonication buffer (1 mM EDTA, 20 mM Tris–HCl pH 7.5) containing 1% protease inhibitor cocktail (P8340, Sigma-Aldrich). The resulting cell suspension was lysed by sonication for 10 x 2 s on ice water bath. Protein content of the sonicates was measured using bicinchoninic acid protein assay by Pierce Biotechnology (Rockford, IL, USA).

Sonicates (50 µg protein/lane) were resolved in 10% SDS–PAGE and blotted onto PVDF membrane (Immobilon-PSQ Transfer Membrane, 0.2 µM pore size, Millipore Corporation, Billerica, MA, USA). Non-specific binding was blocked by 5% non-fat dry milk in Tris-buffered saline containing 0.1% of Tween 20 (TTBS). Blots were incubated overnight in primary antibody [1:500 Mouse anti-G{alpha}i-2 monoclonal antibody, MAB3077 (Chemicon International, Temecula, CA, USA)] in 2.5% non-fat dry milk in TTBS at +4°C. After 4 x 10 min washes in TTBS, the blots were incubated in secondary antibody [1:2500 Goat anti-mouse IgG-HRP, A2554 (Sigma-Aldrich)] in 2.5% non-fat dry milk in TTBS for 1 h at 20°C. Blots were washed 4 x 10 min with TTBS and the chemiluminescent reaction was started with Western Lightning ECL substrate (Perkin-Elmer). The signal was detected on Hyperfilm ECL (Cat. no. RPN3103K, GE Healthcare UK Ltd, Buckinghamshire, UK). The films were scanned using ImageScanner (GE Healthcare UK Ltd).

Experimental design
PCR was used to study the expression of different target genes in OE-E6/E7 cell line. In order to clarify the effects of sex hormones on gene expression, OE-E6/E7 cell line was first cultured with different hormone concentrations for 5 days. Q-PCR was then performed using cDNA of the hormone-treated cells.

Hormone concentrations used in the present study were selected based on the reference serum levels for human females. For estradiol, normal physiological serum levels span from 0.1–1 nmol/l (during the menstrual cycle) to 10–100 nmol/l (during pregnancy). For progesterone, physiological serum concentrations during menstrual cycle vary from 1–10 nmol/l (follicular phase and at ovulation) to 10–100 nmol/l (luteal phase). Additionally, toward the end of pregnancy and parturition, up to 1000 nmol/l, progesterone concentrations are relevant, and were thus included in this study.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Funding
 Acknowledgements
 References
 
PCR revealed the expression of G{alpha}i2 gene and Western blot the presence of the G{alpha}i2 protein in an immortalized human oviductal cell line
Our PCR studies confirmed that G{alpha}i2 gene is expressed in the immortalized human oviductal cell line OE-E6/E7 (Fig. 1A). Control experiments with non-reverse transcribed RNA of each sample confirmed that there was no contamination of human DNA in the samples. To verify the presence of G{alpha}i2 protein in OE-E6/E7 cells, Western blot analysis was carried out. Our results confirmed that G{alpha}i2 is present, although at a relatively low level, in these cells (Fig. 1B).


Figure 1
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  		Figure 1: Expression of G{alpha}i2 gene and presence of G{alpha}i2 protein was confirmed in OE-E6/E7 cell line (A) PCR showed G{alpha}i2 expression in an immortalized Fallopian tube epithelial cell line (OE-E6/E7). PCR products were separated on 1.2% agarose gel. 1, β-actin (643 bp); 2, β actin RT control; 3, G{alpha}i2 (212 bp); 4, G{alpha}i2 RT control, MW, molecular weight (bp). (B) Western blot analysis confirmed the presence of G{alpha}i2 protein in immortalized Fallopian tube epithelial cell line (OE-E6/E7). 1, G protein standard, (2 µl / lane) bovine brain immunoblot standard (Calbiochem); 2, sonicate of Fallopian tube epithelial cells (50 µg/lane)

 
Hormone treatment revealed up-regulation of G{alpha}i2 gene by estradiol
In order to evaluate the effects of steroid hormones on expression of G{alpha}i2 gene, a hormone treatment experiment was carried out using OE-E6/E7 cell line. The cells were treated for 5 days with four concentrations of estradiol (0.1, 1, 10 and 100 nM) and progesterone (1, 10, 100 and 1000 nM), spanning the physiological concentration range of these hormones in human in vivo. Q-PCR revealed that G{alpha}i2 gene was up-regulated by low concentrations of estradiol (Fig. 2A). Cells treated with progesterone did not show any statistically significant changes in G{alpha}i2 expression (Fig. 2B).


Figure 2
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  		Figure 2: Q-PCR showed regulatory effects of estradiol and progesterone on expression of G{alpha}i2 gene

Immortalized Fallopian tube epithelial cell line (OE-E6/E7) was treated in triplicates with either estradiol (A) or progesterone (B) using concentrations of 0.1, 1, 10 and 100 nM or 1, 10, 100 and 1000 nM, respectively. The figure illustrates mean ± SEM of normalized G{alpha}i2 gene expression (n=9). Expression of G{alpha}i2 gene after estrogen treatment at 0.1 nM is significantly higher than after control treatment. *P < 0.05; One-way ANOVA with Tukey’s multiple comparison test

 
The potential nuclear target receptors for sex hormones were determined by PCR
In an attempt to clarify the potential receptor targets for sex hormones in OE-E6/E7 cells, we carried out PCR analysis using primers targeted against various sex hormone receptors (Table I). Our results show that in OE-E6/E7 cell line, only one nuclear receptor target for estrogen is present. Our data showed that ERβ receptor is expressed in these cells (Fig. 3B). No ER{alpha} receptor expression was detected (Fig. 3A). To verify this finding, the functionality of ER{alpha} primer used for PCR was also tested using cDNA of human Fallopian tube tissue and endometrium, and in both of these tissues ER{alpha} was expressed (Fig. 3A).


Figure 3
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  		Figure 3: Expression of estrogen receptors was studied in an immortalized Fallopian tube epithelial cell line (OE-E6/E7)

For control purposes, we also used human Fallopian tube tissue and human endometrium tissue cDNA, since no ER{alpha} expression was seen in OE-E6/E7 cell line. ER{alpha} (A) 1, β-actin (643 bp) in OE-E6/E7 cell line; 2, RT control for β-actin in OE-E6/E7 cell line; 3, ER{alpha} (288 bp) in human Fallopian tube tissue; 4, RT control for ER{alpha} in human Fallopian tube; 5, ER{alpha} (288 bp) in human endometrium; 6, RT control for ER{alpha} in human endometrium; 7, ER{alpha} in OE-E6/E7 cell line; 8, RT control for ER{alpha} in OE-E6/E7 cell line. ERβ in OE-E6/E7 cell line (B), 1, β-actin (643 bp); 2, β-actin RT control; 3, ERβ (217 bp); 4, ERβ RT control. MW, molecular weight (bp). PCR products were separated on 1.2% agarose gel

 
For progesterone, several receptor targets are expressed in OE-E6/E7 cell line (Fig. 4). Our studies showed that the novel membrane receptors for progesterone, namely mPR{alpha} and mPRβ, were both found to be expressed in OE-E6/E7 cell line. However, the expression level of mPRβ was very low. Additionally, nuclear PR-B receptor subtype was found to be expressed in these cells, as well as a PCR product common to PR-A and PR-B subtypes. The expression level of both PR-A,B and PR-B was very similar. For each of the receptors studied, control experiments with non-reverse transcribed RNA of each sample confirmed that there was no contamination of human DNA in the samples.


Figure 4
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  		Figure 4: PCR showed expression of different progesterone receptors in an immortalized Fallopian tube epithelial cell line (OE-E6/E7)

mPR{alpha} (A), 1, β-actin (643 bp); 2, β-actin RT control; 3, mPR{alpha} (200 bp); 4, mPR{alpha} RT control. mPRβ (B), 1, β-actin (643 bp); 2, β-actin RT control; 3, mPRβ (289 bp); 4, mPRβ RT control. PR-A,B (C), 1, β-actin (643 bp); 2, β-actin RT control; 3, PR-A,B (221 bp); 4, PR-A,B RT control. PR-B (D), 1, β-actin (643 bp); 2, β-actin RT control; 3, PR-B (232 bp); 4, PR-B RT control. MW: molecular weight (bp). PCR products were separated on 1.2% agarose gel

 
The novel, G protein-coupled membrane receptor mPR{alpha} was down-regulated by estradiol
Since we found estradiol to up-regulate G{alpha}i2, we aimed at investigating the potential effects of sex hormones on the novel, G protein-coupled membrane receptors for progesterone, which could represent potential signalling partners for G{alpha}i2 in human reproductive tissues. We carried out Q-PCR on hormone-treated OE-E6/E7 cell line cDNA. Our data showed that mPR{alpha} expression is down-regulated at estradiol concentration of 1 nM whereas other concentrations show no changes compared with control (Fig. 5A). Down-regulation of mPR{alpha} roughly coincided with G{alpha}i2 up-regulation observed in this study. We also studied the effects of progesterone on mPR{alpha} gene expression and found that mPR{alpha} expression is up-regulated by 100 nM progesterone treatment (Fig. 5B). Owing to relatively low expression level of the other sex hormone receptors present in OE-E6/E7 cell line, we were unable to investigate the regulation of these genes following estradiol or progesterone treatment.


Figure 5
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  		Figure 5: Q-PCR showed regulatory effects of estradiol and progesterone on expression of mPR{alpha} gene

Immortalized Fallopian tube epithelial cell line (OE-E6/E7) was treated in triplicates with either estradiol (A) or progesterone (B) using concentrations of 0.1, 1, 10 and 100 nM or 1, 10, 100 and 1000 nM, respectively. The figure illustrates mean ± SEM of normalized mPR{alpha} gene expression (n = 9). Expression of mPR{alpha} gene after 1 nM estradiol treatment is significantly lower and after 100 nM progesterone treatment significantly higher than after control treatments. *P < 0.05; ***P < 0.001; One-way ANOVA with Tukey’s multiple comparison test

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Funding
 Acknowledgements
 References
 
The present study demonstrates that low concentrations of estradiol up-regulate the expression of G{alpha}i2 gene in immortalized human oviductal epithelial cell line OE-E6/E7, whereas progesterone has no significant effect. In contrast, our data show membrane progesterone receptor mPR{alpha} gene to be down-regulated by 1 nM estradiol and up-regulated by 100 nM progesterone. It seems sex hormones regulate the expression of G{alpha}i2 and mPR{alpha} genes in a reverse manner in OE-E6/E7 cells. Our results suggest that estrogen receptor ERβ mediates regulatory effects of estradiol in these cells.

Earlier, the presence and role of G{alpha}i2 in myometrium has been intensively studied (Milligan et al., 1989; Tanfin et al., 1991; Europe-Finner et al., 1993; Cohen-Tannoudji et al., 1995), and the role of G{alpha}i2 in the maintenance of myometrium relaxation during pregnancy has been established (Mhaouty et al., 1995). Apart from our recent study proposing hormonal regulation for G{alpha}i2 in human endometrium in vivo (Mönkkönen et al., 2007a), there are few reports indicative of the presence of Gi family proteins in human endometrium (Bernardini et al., 1995, 1999). Prior to the present study, however, the individual roles of estradiol and progesterone in regulation of G{alpha}i2 in female reproductive tract have remained unexplored. Since our previous study (Mönkkönen et al., 2007a) showed the increase in G{alpha}i2 gene expression under the changing physiological hormone combinations during the menstrual cycle, the present study aimed to clarify the potential individual roles of estradiol and progesterone in regulation of selected signalling components such as G{alpha}i2. Future studies should be targeted toward understanding the synergistic or inhibitory effect of different combinations of sex hormones on expression and function of G{alpha}i2.

Previous studies on rat myometrium have demonstrated that estradiol administration increases the levels of both G{alpha}i2 protein and G{alpha}i2 mRNA during pregnancy (Cohen-Tannoudji et al., 1995). These findings are in line with our present data on estradiol-induced up-regulation of G{alpha}i2 gene in immortalized human oviductal epithelial cell line. Regarding our recent studies showing significant increase in G{alpha}i2 gene expression in human endometrium during the menstrual cycle (Mönkkönen et al., 2007a), it seems likely that the increase observed then might be resulting from concentration peak of estrogen at late proliferative phase. It would be reasonable to confirm our present results of G{alpha}i2 gene up-regulation by studying the levels of G{alpha}i2 protein in OE-E6/E7 cells before and after hormone treatment. However, since both the mRNA and protein levels of G{alpha}i2 are relatively modest in OE-E6/E7 cells, it is probable that immunoblotting would not be sensitive enough to detect the differences. Previously, progesterone treatment has been reported to have no effect on G{alpha}i2 expression in rat myometrium during pregnancy (Cohen-Tannoudji et al., 1995). This is in line with our data, since in the present study, progesterone treatment did not cause any significant differences in G{alpha}i2 expression.

Nuclear receptor for estrogen, ER{alpha} (Green et al., 1986), was long considered as the only estrogen receptor and thus responsible for all estrogen actions. Since the discovery of the second nuclear estrogen receptor ERβ (Kuiper et al., 1996; Mosselman et al., 1996), it has become evident that ER{alpha} and ERβ not only have separate genes to encode them, but also distinct functions and transcriptional activity (Byers et al., 1997; Weihua et al., 2000; Critchley et al., 2001). In the rat oviduct epithelium, only ER{alpha} subtype is expressed (Fitzpatrick et al., 1999; Sar and Welsch, 1999; Pelletier et al., 2000), whereas immunolocalization studies in human have demonstrated both estrogen receptors ER{alpha} and ERβ to be present in Fallopian tube epithelial cells (Taylor and Al-Azzawi, 2000). Our present data indicate that ERβ is the only nuclear receptor for estrogen expressed in OE-E6/E7 cell line. In our previous studies, we also noted limited ER{alpha} gene expression, but were unable to detect ER{alpha} protein presence (Lee et al., 2001). Probably ER{alpha} transcript has been lost during the passage of these cells, since presently only ERβ is expressed in OE-E6/E7 cell line. This finding suggests that ERβ is likely to mediate the hormonal regulation of G{alpha}i2 and mPR{alpha}, which was observed in our study. However, given the complexity of estrogen signalling and its modulation in vivo, direct conclusions regarding ERβ-mediated regulation of G{alpha}i2 and mPR{alpha} genes in human in vivo cannot be drawn based on our data. During the recent years, evaluation of estrogen signalling pathways has become more complicated than ever, as completely novel aspects on estrogen signalling have emerged. A number of recent reports have provided evidence of functional G protein-coupled receptors for estrogen (Benten et al., 2001; Qiu et al., 2003; Maggiolini et al., 2004; Revankar et al., 2005; Thomas et al., 2005; Vivacqua et al., 2006). Some of these report receptor localization on plasma membrane (Thomas et al., 2005) and some, unexpectedly, in endoplasmic reticulum (Revankar et al., 2005). Clearly, more studies are needed to clarify the role of these novel classes of estrogen receptors in reproductive physiology.

In this study, we investigated which of the known progesterone receptors are expressed in OE-E6/E7 cell line and found that both membrane receptors mPR{alpha} and mPRβ are expressed, although the expression level of mPRβ was relatively low. Additionally, we detected expression for nuclear progesterone receptors PR-A,B and PR-B, at similar expression level for both. Both PR-A and PCR-B are products of a single gene, and the only difference between them is the additional 165 amino acids present solely in the N-terminus of PR-B receptor. Since the expression level of PR-B was similar to that obtained with the primer pair recognizing both PR-A and PR-B receptor subtypes, we concluded that PR-B is the main nuclear PR receptor in these cells, and thus, might be responsible for the progesterone-induced genomic functions, namely mPR{alpha} up-regulation, observed in this study.

To date, membrane progesterone receptors have been characterized in various species, including human (Zhu et al., 2003a,b). A recent study established the presence and functionality of mPR{alpha} and mPRβ in human myometrium and used human myometrial cells to study their hormonal regulation. Both mPR{alpha} and mPRβ were reported to be up-regulated with 100 nM estradiol, while mPR{alpha} was also up-regulated with 100 nM progesterone and estradiol-progesterone combination (Karteris et al., 2006). Regarding our present results in the light of these previous findings, our data on 100 nM progesterone-induced mPR{alpha} up-regulation are in line with Karteris et al. results. However, with estradiol we observed down-regulation of mPR{alpha} at 1 nM, and no effect on mPR{alpha} expression at 100 nM, the concentration at which Karteris et al. reported up-regulation. Potential factors behind these differences could be either the different cell types used and thus variable repertoire of transcriptional factors or, alternatively, differences in estradiol concentrations and treatment times. Namely, Karteris et al. used acute, 4–16 h treatments, whereas our cell line was cultured with hormones for 5 days. In the present study, we investigated only hormonal regulation of mPR{alpha}, since the expression level of mPRβ in OE-E6/E7 cell line was too low for delicate quantitative genomic analyses.

Earlier studies have shown that Gi family proteins have a role in the signal transduction of rapid, non-genomic actions of estrogen (Zhu et al., 2003b) and progesterone (Karteris et al., 2006). Given the established relationship between Gi family proteins and the G protein-coupled membrane receptors for progesterone, it is relevant to assume that G{alpha}i2 and progesterone receptor mPR{alpha} might have a common signalling pathway in Fallopian tube epithelial cells. However, establishing the direct signalling relationship between the two would require future experimentation. Progesterone is known to decrease ciliary motility in Fallopian tube (Mahmood et al., 1998), and thus have a regulatory role in gamete transport. Recent studies have shown the specific localization of nuclear progesterone receptors (Teilmann et al., 2006) and the novel mPR{gamma} receptors (Nutu et al., 2007) in oviductal luminal epithelial cells and the ciliary membrane in rat and human, and proposed a role for those receptors in ciliary beat regulation and gamete transport. Earlier, a rapid, non-genomic signalling pathway for progesterone has been proposed in regulation of ciliary motility (Wessel et al., 2004).

Immortalized human oviductal cell line OE-E6/E7 used in our study has been shown to retain morphological features characteristic of secretory epithelial cells (Ling et al., 2005). Although many signalling events in Fallopian tube epithelial cells are likely to require contribution of secretory cells, lack of ciliated cells in this cell line is naturally limiting the interpretation of the results. Since G{alpha}i2 is, in various tissues, localized specifically in cells which have motile cilia (Shinohara et al., 1998; Ostrowski et al., 2002; Mönkkönen et al., 2007a), it is likely to have a general, cilia-specific role. As a first proof of this, we recently showed G{alpha}i2 to control ciliary motility and cerebrospinal fluid homeostasis in rat brain (Mönkkönen et al., 2007b).

Regarding other potential roles for G{alpha}i2 in Fallopian tube epithelial cells, it is noteworthy that recently, a Toll-like receptor (TLR) signalling regulating, anti-inflammatory role has been proposed for G{alpha}i2 by an yet unknown mechanism (Fan et al., 2005). According to our previous studies, localization pattern of G{alpha}i2 in human reproductive tract (Mönkkönen et al., 2007a) is similar to that of several TLRs in female reproductive tract (Fazeli et al., 2005). Interestingly, various TLRs also seem to have hormonal regulation similar to G{alpha}i2 in human endometrium in vivo (Aflatoonian et al., 2007).

In conclusion, our study demonstrates for the first time the hormonal regulation of G{alpha}i2 expression in human oviductal epithelial cell line OE-E6/E7 by estradiol. Additionally, the present study provides valuable information on hormonal regulation of the novel, G protein-coupled membrane receptor for progesterone, mPR{alpha}, which was shown to be up-regulated by progesterone and down-regulated by estradiol in OE-E6/E7 cell line. Furthermore, our study suggested ERβ receptor to mediate the estradiol-induced regulation of G{alpha}i2 and mPR{alpha} expression.


   Funding
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Funding
Acknowledgements
 References
 
The Association of Finnish Pharmacies; Finnish Pharmaceutical Society; Alfred Kordelin Foundation; Farmos Science and Research Foundation; Helsingin Sanomat Centennial Foundation; The Saastamoinen Foundation.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Funding
 Acknowledgements
References
 
We wish to thank Ms Elizabeth M. Tuckerman and Prof T.C. Li for providing human endometrial biopsies and Fallopian tube tissue for cDNA synthesis. Ms Chris Bruce and Ms Sarah Elliott are acknowledged for skilful technical assistance.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Funding
 Acknowledgements
 References
 
Aflatoonian R, Tuckerman E, Elliott SL, Bruce C, Aflatoonian A, Li TC, Fazeli A. Menstrual cycle-dependent changes of Toll-like receptors in endometrium. Hum Reprod (2007) 22:586–593.[Abstract/Free Full Text]

Arey BJ, Stevis PE, Deecher DC, Shen ES, Frail DE, Negro-Vilar A, Lopez FJ. Induction of promiscuous G protein coupling of the follicle-stimulating hormone (FSH) receptor: a novel mechanism for transducing pleiotropic actions of FSH isoforms. Mol Endocrinol (1997) 11:517–526.[Abstract/Free Full Text]

Arias EB, Verhage HG, Jaffe RC. Complementary deoxyribonucleic acid cloning and molecular characterization of an estrogen-dependent human oviductal glycoprotein. Biol Reprod (1994) 51:685–694.[Abstract]

Baillie HS, Pacey AA, Warren MA, Scudamore IW, Barratt CL. Greater numbers of human spermatozoa associate with endosalpingeal cells derived from the isthmus compared with those from the ampulla. Hum Reprod (1997) 12:1985–1992.[Abstract/Free Full Text]

Benten WP, Stephan C, Lieberherr M, Wunderlich F. Estradiol signaling via sequestrable surface receptors. Endocrinology (2001) 142:1669–1677.[Abstract/Free Full Text]

Bernardini L, Moretti-Rojas I, Brush M, Rojas FJ, Balmaceda JP. Status of hCG/LH receptor and G proteins in human endometrium during artificial cycles of hormone replacement therapy. J Soc Gynecol Investig (1995) 2:630–635.[CrossRef][Web of Science][Medline]

Bernardini L, Moretti-Rojas I, Brush M, Rojas FJ, Balmaceda JP. Changes in expression of adenyl cyclase activity in human endometrium during hormone replacement therapy and ovarian stimulation. Mol Hum Reprod (1999) 5:955–960.[Abstract/Free Full Text]

Byers M, Kuiper GG, Gustafsson JA, Park-Sarge OK. Estrogen receptor-beta mRNA expression in rat ovary: down-regulation by gonadotropins. Mol Endocrinol (1997) 11:172–182.[Abstract/Free Full Text]

Chi L, Zhou W, Prikhozhan A, Flanagan C, Davidson JS, Golembo M, Illing N, Millar RP, Sealfon SC. Cloning and characterization of the human GnRH receptor. Mol Cell Endocrinol (1993) 91:R1–R6.[CrossRef][Web of Science][Medline]

Cohen-Tannoudji J, Mhaouty S, Elwardy-Merezak J, Lecrivain JL, Robin MT, Legrand C, Maltier JP. Regulation of myometrial Gi2, Gi3, and Gq expression during pregnancy. Effects of progesterone and estradiol. Biol Reprod (1995) 53:55–64.[Abstract]

Critchley HO, Brenner RM, Henderson TA, Williams K, Nayak NR, Slayden OD, Millar MR, Saunders PT. Estrogen receptor beta, but not estrogen receptor alpha, is present in the vascular endothelium of the human and nonhuman primate endometrium. J Clin Endocrinol Metab (2001) 86:1370–1378.[Abstract/Free Full Text]

Donnez J, Casanas-Roux F, Caprasse J, Ferin J, Thomas K. Cyclic changes in ciliation, cell height, and mitotic activity in human tubal epithelium during reproductive life. Fertil Steril (1985) 43:554–559.[Web of Science][Medline]

Europe-Finner GN, Phaneuf S, Watson SP, Lopez Bernal A. Identification and expression of G-proteins in human myometrium: up-regulation of G alpha s in pregnancy. Endocrinology (1993) 132:2484–2490.[Abstract/Free Full Text]

Fan H, Zingarelli B, Peck OM, Teti G, Tempel GE, Halushka PV, Spicher K, Boulay G, Birnbaumer L, Cook JA. Lipopolysaccharide- and gram-positive bacteria-induced cellular inflammatory responses: role of heterotrimeric Galpha(i) proteins. Am J Physiol Cell Physiol (2005) 289:C293–C301.[Abstract/Free Full Text]

Fazeli A, Bruce C, Anumba DO. Characterization of toll-like receptors in the female reproductive tract in humans. Hum Reprod (2005) 20:1372–1378.[Abstract/Free Full Text]

Fitzpatrick SL, Funkhouser JM, Sindoni DM, Stevis PE, Deecher DC, Bapat AR, Merchenthaler I, Frail DE. Expression of estrogen receptor-beta protein in rodent ovary. Endocrinology (1999) 140:2581–2591.[Abstract/Free Full Text]

Green S, Walter P, Kumar V, Krust A, Bornert JM, Argos P, Chambon P. Human oestrogen receptor cDNA: sequence, expression and homology to v-erb-A. Nature (1986) 320:134–139.[CrossRef][Medline]

Hawes BE, Barnes S, Conn PM. Cholera toxin and pertussis toxin provoke differential effects on luteinizing hormone release, inositol phosphate production, and gonadotropin-releasing hormone (GnRH) receptor binding in the gonadotrope: evidence for multiple guanyl nucleotide binding proteins in GnRH action. Endocrinology (1993) 132:2124–2130.[Abstract/Free Full Text]

Hepler JR, Gilman AG. G proteins. Trends Biochem Sci (1992) 17:383–387.[CrossRef][Web of Science][Medline]

Herrlich A, Kuhn B, Grosse R, Schmid A, Schultz G, Gudermann T. Involvement of Gs and Gi proteins in dual coupling of the luteinizing hormone receptor to adenylyl cyclase and phospholipase C. J Biol Chem (1996) 271:16764–16772.[Abstract/Free Full Text]

Jansen RP. Endocrine response in the Fallopian tube. Endocr Rev (1984) 5:525–551.[Abstract/Free Full Text]

Karteris E, Zervou S, Pang Y, Dong J, Hillhouse EW, Randeva HS, Thomas P. Progesterone signaling in human myometrium through two novel membrane G protein-coupled receptors: potential role in functional progesterone withdrawal at term. Mol Endocrinol (2006) 20:1519–1534.[Abstract/Free Full Text]

Kervancioglu ME, Djahanbakhch O, Aitken RJ. Epithelial cell coculture and the induction of sperm capacitation. Fertil Steril (1994) 61:1103–1108.[Web of Science][Medline]

Kervancioglu ME, Saridogan E, Aitken RJ, Djahanbakhch O. Importance of sperm-to-epithelial cell contact for the capacitation of human spermatozoa in Fallopian tube epithelial cell cocultures. Fertil Steril (2000) 74:780–784.[CrossRef][Web of Science][Medline]

Krsmanovic LZ, Mores N, Navarro CE, Tomic M, Catt KJ. Regulation of Ca2+-sensitive adenylyl cyclase in gonadotropin-releasing hormone neurons. Mol Endocrinol (2001) 15:429–440.[Abstract/Free Full Text]

Krsmanovic LZ, Mores N, Navarro CE, Arora KK, Catt KJ. An agonist-induced switch in G protein coupling of the gonadotropin-releasing hormone receptor regulates pulsatile neuropeptide secretion. Proc Natl Acad Sci USA (2003) 100:2969–2974.[Abstract/Free Full Text]

Kuiper GG, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson JA. Cloning of a novel receptor expressed in rat prostate and ovary. Proc Natl Acad Sci USA (1996) 93:5925–5930.[Abstract/Free Full Text]

Lee YL, Lee KF, Xu JS, Wang YL, Tsao SW, Yeung WS. Establishment and characterization of an immortalized human oviductal cell line. Mol Reprod Dev (2001) 59:400–409.[CrossRef][Web of Science][Medline]

Ling L, Lee YL, Lee KF, Tsao SW, Yeung WS, Kan FW. Expression of human oviductin in an immortalized human oviductal cell line. Fertil Steril (2005) 84(Suppl_2):1095–1103.[CrossRef][Web of Science][Medline]

Liu LP, Chan ST, Ho PC, Yeung WS. Human oviductal cells produce high molecular weight factor(s) that improves the development of mouse embryo. Hum Reprod (1995) 10:2781–2786.[Abstract/Free Full Text]

Loosfelt H, Misrahi M, Atger M, Salesse R, Vu Hai-Luu Thi MT, Jolivet A, Guiochon-Mantel A, Sar S, Jallal B, Garnier J, et al. Cloning and sequencing of porcine LH-hCG receptor cDNA: variants lacking transmembrane domain. Science (1989) 245:525–528.[Abstract/Free Full Text]

Maggiolini M, Vivacqua A, Fasanella G, Recchia AG, Sisci D, Pezzi V, Montanaro D, Musti AM, Picard D, Ando S. The G protein-coupled receptor GPR30 mediates c-fos up-regulation by 17beta-estradiol and phytoestrogens in breast cancer cells. J Biol Chem (2004) 279:27008–27016.[Abstract/Free Full Text]

Mahmood T, Saridogan E, Smutna S, Habib AM, Djahanbakhch O. The effect of ovarian steroids on epithelial ciliary beat frequency in the human Fallopian tube. Hum Reprod (1998) 13:2991–2994.[Abstract/Free Full Text]

McFarland KC, Sprengel R, Phillips HS, Kohler M, Rosemblit N, Nikolics K, Segaloff DL, Seeburg PH. Lutropin–choriogonadotropin receptor: an unusual member of the G protein-coupled receptor family. Science (1989) 245:494–499.[Abstract/Free Full Text]

Mhaouty S, Cohen-Tannoudji J, Bouet-Alard R, Limon-Boulez I, Maltier JP, Legrand C. Characteristics of the alpha 2/beta 2-adrenergic receptor-coupled adenylyl cyclase system in rat myometrium during pregnancy. J Biol Chem (1995) 270:11012–11016.[Abstract/Free Full Text]

Milligan G, Tanfin Z, Goureau O, Unson C, Harbon S. Identification of both Gi2 and a novel, immunologically distinct, form of Go in rat myometrial membranes. FEBS Lett (1989) 244:411–416.[CrossRef][Web of Science][Medline]

Minegishi T, Nakamura K, Takakura Y, Miyamoto K, Hasegawa Y, Ibuki Y, Igarashi M, Minegish T. Cloning and sequencing of human LH/hCG receptor cDNA. Biochem Biophys Res Commun (1990) 172:1049–1054.[CrossRef][Web of Science][Medline]

Minegishi T, Nakamura K, Takakura Y, Ibuki Y, Igarashi M, Minegish T. Cloning and sequencing of human FSH receptor cDNA. Biochem Biophys Res Commun (1991) 175:1125–1130.[CrossRef][Web of Science][Medline]

Mönkkönen KS, Aflatoonian R, Lee KF, Yeung WS, Tsao SW, Laitinen JT, Tuckerman EM, Li TC, Fazeli A. Localization and variable expression of G{alpha}i2 in human endometrium and Fallopian tubes. Hum Reprod (2007) a 22:1224–1230.[Abstract/Free Full Text]

Mönkkönen KS, Hakumäki JM, Hirst RA, Miettinen RA, O’Callaghan C, Männistö PT, Laitinen JT. Intracerebroventricular antisense knockdown of G alpha i2 results in ciliary stasis and ventricular dilatation in the rat. BMC Neurosci (2007) b 8:26.[CrossRef][Medline]

Mosselman S, Polman J, Dijkema R. ER beta: identification and characterization of a novel human estrogen receptor. FEBS Lett (1996) 392:49–53.[CrossRef][Web of Science][Medline]

Murray SC, Smith TT. Sperm interaction with Fallopian tube apical membrane enhances sperm motility and delays capacitation. Fertil Steril (1997) 68:351–357.[CrossRef][Web of Science][Medline]

Nutu M, Weijdegard B, Thomas P, Bergh C, Thurin-Kjellberg A, Pang Y, Billig H, Larsson DG. Membrane progesterone receptor gamma: tissue distribution and expression in ciliated cells in the Fallopian tube. Mol Reprod Dev (2007) 74:843–850.[CrossRef][Web of Science][Medline]

Ostrowski LE, Blackburn K, Radde KM, Moyer MB, Schlatzer DM, Moseley A, Boucher RC. A proteomic analysis of human cilia: identification of novel components. Mol Cell Proteomics (2002) 1:451–465.[Abstract/Free Full Text]

Pacey AA, Hill CJ, Scudamore IW, Warren MA, Barratt CL, Cooke ID. The interaction in vitro of human spermatozoa with epithelial cells from the human uterine (Fallopian) tube. Hum Reprod (1995) 10:360–366.[Abstract/Free Full Text]

Pelletier G, Labrie C, Labrie F. Localization of oestrogen receptor alpha, oestrogen receptor beta and androgen receptors in the rat reproductive organs. J Endocrinol (2000) 165:359–370.[Abstract]

Qiu J, Bosch MA, Tobias SC, Grandy DK, Scanlan TS, Ronnekleiv OK, Kelly MJ. Rapid signaling of estrogen in hypothalamic neurons involves a novel G-protein-coupled estrogen receptor that activates protein kinase C. J Neurosci (2003) 23:9529–9540.[Abstract/Free Full Text]

Revankar CM, Cimino DF, Sklar LA, Arterburn JB, Prossnitz ER. A transmembrane intracellular estrogen receptor mediates rapid cell signaling. Science (2005) 307:1625–1630.[Abstract/Free Full Text]

Sar M, Welsch F. Differential expression of estrogen receptor-beta and estrogen receptor-alpha in the rat ovary. Endocrinology (1999) 140:963–971.[Abstract/Free Full Text]

Shinohara H, Asano T, Kato K, Kameshima T, Semba R. Localization of a G protein Gi2 in the cilia of rat ependyma, oviduct and trachea. Eur J Neurosci (1998) 10:699–707.[CrossRef][Web of Science][Medline]

Sprengel R, Braun T, Nikolics K, Segaloff DL, Seeburg PH. The testicular receptor for follicle stimulating hormone: structure and functional expression of cloned cDNA. Mol Endocrinol (1990) 4:525–530.[Abstract/Free Full Text]

Stanislaus D, Ponder S, Ji TH, Conn PM. Gonadotropin-releasing hormone receptor couples to multiple G proteins in rat gonadotrophs and in GGH3 cells: evidence from palmitoylation and overexpression of G proteins. Biol Reprod (1998) 59:579–586.[Abstract/Free Full Text]

Tanfin Z, Goureau O, Milligan G, Harbon S. Characterization of G proteins in rat myometrium. A differential modulation of Gi2 alpha and Gi3 alpha during gestation. FEBS Lett (1991) 278:4–8.[CrossRef][Web of Science][Medline]

Taylor AH, Al-Azzawi F. Immunolocalisation of oestrogen receptor beta in human tissues. J Mol Endocrinol (2000) 24:145–155.[Abstract]

Teilmann SC, Clement CA, Thorup J, Byskov AG, Christensen ST. Expression and localization of the progesterone receptor in mouse and human reproductive organs. J Endocrinol (2006) 191:525–535.[Abstract/Free Full Text]

Thomas P, Pang Y, Filardo EJ, Dong J. Identity of an estrogen membrane receptor coupled to a G protein in human breast cancer cells. Endocrinology (2005) 146:624–632.[Web of Science][Medline]

Tsutsumi M, Zhou W, Millar RP, Mellon PL, Roberts JL, Flanagan CA, Dong K, Gillo B, Sealfon SC. Cloning and functional expression of a mouse gonadotropin-releasing hormone receptor. Mol Endocrinol (1992) 6:1163–1169.[Abstract/Free Full Text]

Verhage HG, Bareither ML, Jaffe RC, Akbar M. Cyclic changes in ciliation, secretion and cell height of the oviductal epithelium in women. Am J Anat (1979) 156:505–521.[CrossRef][Web of Science][Medline]

Verhage HG, Fazleabas AT, Donnelly K. The in vitro synthesis and release of proteins by the human oviduct. Endocrinology (1988) 122:1639–1645.[Abstract/Free Full Text]

Vivacqua A, Bonofiglio D, Recchia AG, Musti AM, Picard D, Ando S, Maggiolini M. The G protein-coupled receptor GPR30 mediates the proliferative effects induced by 17beta-estradiol and hydroxytamoxifen in endometrial cancer cells. Mol Endocrinol (2006) 20:631–646.[Abstract/Free Full Text]

Wagh PV, Lippes J. Human oviductal fluid proteins. III. Identification and partial purification. Fertil Steril (1989) 51:81–88.[Web of Science][Medline]

Weihua Z, Saji S, Makinen S, Cheng G, Jensen EV, Warner M, Gustafsson JA. Estrogen receptor (ER) beta, a modulator of ERalpha in the uterus. Proc Natl Acad Sci USA (2000) 97:5936–5941.[Abstract/Free Full Text]

Wessel T, Schuchter U, Walt H. Ciliary motility in bovine oviducts for sensing rapid non-genomic reactions upon exposure to progesterone. Horm Metab Res (2004) 36:136–141.[CrossRef][Web of Science][Medline]

Yeung WS, Lau EY, Chan ST, Ho PC. Coculture with homologous oviductal cells improved the implantation of human embryos—a prospective randomized control trial. J Assist Reprod Genet (1996) 13:762–767.[CrossRef][Web of Science][Medline]

Zhu Y, Bond J, Thomas P. Identification, classification, and partial characterization of genes in humans and other vertebrates homologous to a fish membrane progestin receptor. Proc Natl Acad Sci USA (2003) a 100:2237–2242.[Abstract/Free Full Text]

Zhu Y, Rice CD, Pang Y, Pace M, Thomas P. Cloning, expression, and characterization of a membrane progestin receptor and evidence it is an intermediary in meiotic maturation of fish oocytes. Proc Natl Acad Sci USA (2003) b 100:2231–2236.[Abstract/Free Full Text]

Submitted on September 2, 2007; accepted on September 24, 2007.


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