Effect of antioestrogens on proliferation , differentiation and telomerase activity of pig granulosa cells in vitro *

The aim of the present work was to study the effect of oestrogen receptor inhibitors (ICI 164.384; ICI and cyclofenil; CF) on proliferation, oestradiol synthesis, aromatase expression and telomerase activity (TA) of pig granulosa cells (GC) derived from small (1-2 mm; SF-GC) and large (5-7 mm; LF-GC) follicles. Cells were treated with anioestrogens for 48 h in basic (medium only) as well as FSH-stimulated conditions. Antioestrogens applied individually and in a combination significantly (P<0.01) decreased proliferative potential of LF-GC. Antioestrogens applied in a combination increased (P<0.05-0.01) 17-β oestradiol synthesis in small and large follicle GC. Antioestrogens applied individually and in a combination caused an increase (P<0.01) of aromatase gene expression in FSH-stimulated conditions as well as in basic conditions. Moreover, antioestrogens increased (P<0.05-0.01) TA in basic and in FSH-stimulated conditions. The results of the study indicate the involvement of oestrogen receptor α and β in the control of proliferation, differentiation and telomerase activity in pig GC. Furthermore, telomerase activity does not have to be linked only to pig GC proliferation but may also be involved in the differentiation process.


INTRODUCTION
Oestrogens are known to be important factors controlling proliferation and differentiation of granulosa cells (Drummond and Findlay, 1999).Oestrogens exert their role by two subtypes of nuclear receptors: ERα and ERβ.Both subtypes of ER have been localized in mammalian ovary.Tomanek et al. (1997) localized protein as well as mRNA of ERα in the sheep ovary.High amount of ERβ protein and mRNA was detected in the granulosa cells of primary, secondary and antral follicles in rodents (Tremblay et al., 1997;Sar et al., 1999) and humans (Enmark et al., 1997;Pelletier et al., 2000).On the other hand low amounts of mRNA and protein of ERα (Pelletier et al., 2000;Saunders et al., 2000) were detected in human ovary.Slomczynska and Wozniak (2001) and Slomczynska et al. (2001) localized oestrogen receptor α and β in the pig ovary with ERβ being predominant.
ERβ was present at all stages of follicular development whereas ERα mRNA and protein were detected only in preovulatory follicles and early corpora lutea.
The effect of oestrogens acting through their nuclear receptors on granulosa cell proliferation and follicular growth is still not fully elucidated.Gore-Langton and Daniel (1990) demonstrated an important role of oestrogens in the control of the growth of rat follicles.Antrum formation in vitro in growing preantral follicles was dependent on synergistic action of oestradiol and FSH.It was observed that the proliferative potential of rat granulosa cells in vitro is stimulated by oestradiol and FSH (Bendell and Dorrington, 1991;Bley et al., 1997).Oestradiol applied alone had no effect on the proliferative potential of bovine granulosa cells cultured in vitro (Langhout et al., 1991).On the contrary, the proliferation of pig granulosa cells from small and large follicles decreased as a result of oestradiol stimulation (Ranson et al., 1997).In our previous study (Chronowska et al., 2009), inhibition of oestradiol synthesis with fadrozole resulted in decreased proliferation of pig granulosa cells (GC ) from small and large follicles.
In our previous study (Tomanek et al., 2008), we demostrated the presence of telomerase activity in freshly isolated as well as cultured pig granulosa cells obtained from small (1-2) and large (5-7 mm) antral follicles.It has been evidenced that telomerase activity is under the control of oestrogens in some oestrogen dependent tissues.Misiti et al. (2000) showed a stimulatory effect of oestrogens on induction of TERT (telomerase reverse transcriptase) expression in human ovarian epithelium.Application of oestradiol to female rats prevented decrease of telomerase activity in granulosa cells (Yamagata et al., 2002).In our previous study (Chronowska et al., 2009), inhibition of oestradiol synthesis by fadrozole resulted in decrease of telomerase activity in pig large follicle granulosa cells.However, the role of oestrogens and the mechanism of their action in telomerase activity regulation in pig granulosa cells is still not fully elucidated.
Antioestrogens (oestrogen receptor antagonists) are a group of medications that are used in cancer theraphy.They block the effect of oestrogen on the growth of tumor.Antioestrogens work by binding to oestrogen receptors, blocking oestrogen from binding to these receptors.This also blocks oestrogen from activating genes for specific growth-promoting proteins.ICI 164.384, belonging to the group of pure antioestrogens, is 7α-alkilamid analog of oestradiol.It blocks ER by the inhibition of AF-1 and AF-2 subdomain activity (Pike et al., 2001).Cyclofenil is non-steroid selective modulator of ER β.It exhibits 5 times higher affinity to ER β than to ERα (Muthyala et al., 2003).
As the effect of oestrogens acting through oestrogen receptors on pig granulosa cell proliferation, differentiation and telomerase activity is still largely unknown, in the present study we applied antioestrogens to elucidate the role of oestrogen receptors in the process of pig granulosa cell proliferation, telomerase activity, oestradiol synthesis and aromatase expression.

Isolation of porcine granulosa cells and in vitro culture conditions
Porcine ovaries were collected from local slaughterhouse and transported to the laboratory in a theromo-cointainer filled with phosphate-buffered saline (PBS) within 30 min.In the laboratory, ovaries were placed in sterile PBS supplemented with an antibiotic antimycotic solution (Sigma, UK) for 20 min and then cut into small pieces.Individual follicles of desired size (small antral 1-2 mm and large antral 5-7 mm) were isolated from the ovarian pieces by dissection and splitopened under a stereomicroscope (Leica MZ6, Switzerland) into DMEM/F12 medium (Gibco, BRL) to obtain granulosa cells.Cell suspension was treated with trypsin and DNase (Sigma, USA) to eliminate dead cells.The number of living cells in suspension was estimated by use of 0.25% trypan blue (Sigma, USA) in PBS and by counting in a haematocytometer.Viability of granulosa cells after trypsyn/DNase treatment was 85-90%.Cells were seeded onto 4-or 24well culture microplates Nunclon Delta (Nunc, Denamrk) in a density of 1-1.5 x 10 5 of living cells/well and cultured as a proliferating monolayer in a DMEM/ F12 medium supplemented with an insuline-transferrin-selenium mixture (ITS-X, 1 ml/100 ml) and gentamicin (all Gibco, BRL).Two percent of foetal bovine serum (FBS, Gibco) was added in order to facilitate plating.Cell cultures were performed at 37ºC in a humified atmosphere of 5% CO 2 in a Sanyo MCO-175M incubator.

Hormonal supplements
Granulosa cells were cultured for 48 h in the presence of oestrogen receptor inhibitor ICI 164.384 (7α alkilamid analog of oestradiol; Pharma, Cergy, France) supplemented into the culture medium at a concentration of 10 -7 M. Antioestrogens were added to the medium 24 h after the beginning of the culture.The cyclofenil (ERβ inhibitor; Sigma, USA) was used at a concentration of 10 -7 M. Testosterone (Steraloids Inc., USA) at a concentration of 10 -8 M was supplemented as an aromatase substrate.The FSH (pFSH-I-1 Iod/Bio, AFP 10640D, NIADDK-NIH, Torrance, CA, USA) was used at a concentration of 50 ng/ml to induce steroidogenesis in the granulosa cells.

Cell proliferation assay
To determine the granulosa cell proliferation potential in vitro, the newly synthesized DNA in cell cultures was measured by incorporation of 3 H-thymidine using the technique of TCA precipitation and liquid scintillation counting as we described earlier (Vackova et al., 2003).

Oestradiol synthesis measurement
The level of oestradiol synthesis in granulosa cells in vitro was evaluated by radioimmunoassay of oestradiol 17-β in the culture medium using RIA techniques which are routinely used in the laboratory (described earlier in Petr et al., 1991).The lower detection limit was 0.066 ng per ml.The intra-assay and inter-assay coefficient of variation was 5.92 and 8.87%, respectively.

Telomerase activity assay
Preparation of granulosa cells.In vitro cultured granulosa cells were prepared as follows: at the end of in vitro culture, the culture media from individual culture wells were removed and stored at -20°C for RIA.Cells were washed with a serum free medium and PBS and detached by accutase (Chemicon, USA).The cells were then transferred to Eppendorf tubes and centrifuged at 4°C and 6000 rpm (3500 G) for 15 min.Pelletes of cells were stored at -80°C until use.

TRAP (Telomeric Repeat Amplification Protocol) assay
At the time of the telomerase activity assay, cell lysates from in vitro cultured cells were prepared in a CHAPS buffer.Briefly: cell pellets of cultured granulosa cells (2 × 10 5 ) and the positive control provided in the kit were resuspended with 200 μl of 1X CHAPS Lysis Buffer.The protein concentration was measured spectrophotometrically.A modified protocol based on the TRAPEZE Telomerase Detection Kit (Chemicon, USA) was applied to evaluate telomerase activity.Internal telomerase assay standard and the fluorescent type-specific TS primer AATCCGTCGAGCAGAGTT-6-FAMTM (Applied Biosystems, USA) we used.Telomerase extension and PCR were performed in a Biometra T gradient PCR thermocycler (Biometra, Germany).Telomerase activity was expressed as TPG/ mg protein (Total Product Generated) per milligram of protein.

Real time RT-PCR quantification of the level of P450 aromatase mRNA
Total RNA was isolated from the cultured cells using 6100 Nucleic Acid PrepStation and Total RNA Chemistry (Applied Biosystems (ABI), USA) and quantified by measuring absorbance at 260 nm.Fifty μl of RNA was transcribed into cDNA using the cDNA Archive Kit (ABI).Glyceraldehyde 3-phosphate dehydrogenase was used as a reference gene.Aromatase primers (forward, 5'-CTGACCGTCTGTGCCGATT-3'; reverse, 5'GCGGACCTCCT-3') and MGB probe (TET -AATCACCAAGCACCTGGA) and glyceraldehyde 3-phosphate dehydrogenase primers (forward, 5'-GAGCATCTCCTGACTTCCAGTTTC-3'; reverse, 5'CCTAAGCCCCTCCCCTTCT-3') and MGB probe (VIC -ATCCC AGACCCCC) were designed using the Primer Express 3.0 Program (ABI).Quantification of aromatase was performed using the ABI 7500 Fast Real-Time PCR System.The PCR mix composition for one probe was as follows: 1 μl of cDNA, 500 nM of forward and reverse primers, 250 nM of TaqMan®MGB probe, 5 μl of 1XTaqMan®Fast Universal MasterMix, NoAmpErase®UNG (Applied Biosystems, USA), water to a volume of 10 μl.The PCR reaction was continued for 40 cycles after the initial denaturation at 95°C for 20 s.Each cycle of PCR consisted of 5 s of denaturation at 95°C and 30 s of annealing at 60°C.

Statistical analysis
The data were obtained from 5 experiments with different pools of smalland large-follicle granulosa cells.Each experiment consisted of three repetitions per treatment.The data are presented as means ± SEM.All data were analysed using the SAS Programme (SAS 2001, SAS System for Windows, Release 8.2 (TS2M0), SAS Inst.Inc. Cary, NC).Two-way ANOVA was used to determine the significance of differences of ICI 164.384 and cyclofenil effect applied alone and in combination, on proliferation, oestradiol production, aromatase expression and telomerase activity of small-follicle and large-follicle granulosa cells in vitro.Differences with a probability of P<0.05 were considered significant.

Granulosa cell proliferation.
In FSH-stimulated conditions, antioestrogens applied in a combination decreased (P<0.01)proliferation rate of small and large GC (Figures 1A and 1B, respectively), while cyclofenil applied alone increased (P<0.05)proliferation of small follicle granulosa cells (Figure 1A).Antioestrogens applied individually and in a combination significantly (P<0.01)decreased proliferative potential of LF-GC (Figure 1B).Oestradiol synthesis.ICI 164.384 increased (P<0.05-0.01)oestradiol synthesis in small and large follicle GC in basic as well as FSH-stimulated conditions.Cyclofenil increased (P<0.05-0.01)oestradiol secretion in small follicle GC.Antioestrogens applied in a combination increased (P<0.05-0.01)17-β oestradiol (E2) synthesis in small and large follicle GC (Figures 2A and 2B,respectively).Telomerase activity.Cyclofenil increased (P<0.01)telomerase activity in small follicle GC in FSH-stimulated conditions (Figures 4A).ICI 164.384 applied alone increased (P<0.05)telomerase activity in large follicle GC in basic as well as FSH-stimulated conditions (Figure 4B).Antioestrogens applied in a combination caused increase (P<0.05-0.01) in TA in basic conditions [SF-GC] and in FSHstimulated conditions [LF-GC] (Figures 4A and 4B, respectively).results were recently obtained in prostate stromal cells where up-regulation of aromatase expression by prostaglandin E2 was mediated by oestrogen receptorrelated receptor α (Miao et al., 2010).These differences may be explained by different regulatory mechanisms in both types of cells.
In the present work, antioestrogens caused an increase in telomerase activity in small and large follicle GC.On the contrary, Misiti et al. (2000) observed that oestrogens induced TERT expression and telomerase activity in human ovarian epithelium cells.In the presence of oestradiol, transient expression of ERα but not ERβ significantly increased TERT promoter activity.Similar results were obtained in human primary haematopoietic cells where down-regulation of ERα but not ERβ inhibited oestrogen-stimulated telomerase function (Calado et al., 2009).Oestrogen receptor α was also proved to mediate effect of oestradiol on telomerase activity in human mesenchymal stem cells (Cha et al., 2008).
Interestingly, in the present study we observed that decreased proliferation of GC cultured in the presence of antioestrogens was not accompanied by decreased telomerase activity.On the other hand, elevated oestradiol synthesis caused by antioestrogens action was accompanied by higher telomerase activity.It is widely accepted that level of telomerase activity correlates with proliferative status of the cell (Tanaka et al., 1998).It was not a case in our study, where telomerase activity correlated rather with differentiation status of the cell defined by oestradiol synthesis and aromatase expression.Similar results were earlier obtained in rat.It was shown that telomerase activity in granulosa cells is likely to play significant role for proper follicular development and that the loss of telomerase activity may lead to follicular atresia.The supplementation of oestradiol protected preovulatory follicles from atresia and maintained telomerase activity in the granulosa cells (Yamagata et al., 2002).

CONCLUSIONS
In conclusion, the results of the present study indicate the involvement of oestrogen receptor α and β in the control of proliferation, differentiation and telomerase activity in pig granulosa cells.Moreover, in support of observation of Yamagata et al. (2002), the results of our study indicate that telomerase activity in porcine granulosa cells does not have to be only linked to their proliferation but also may be involved in the control of some differentiation processes.

Figure 1A .Figure 1B .
Figure 1A.Proliferation of porcine granulosa cells derived from small (1-2 mm) follicles assayed by incorporation of 3 H thymidine after 48 h of in vitro culture in the presence of ICI 164.384 (10 -7 M) and cyclofenil (10 -7 M) applied individually and in combination in basic conditions and in FSH (50 ng/ml) stimulated conditions.Each bar represents mean ± SEM for 5 experiments performed in triplicates.* and ** denotes means significantly different from control (Ctrl for basic conditions, FSH for FSH-stimulated conditions; P<0.05-0.01)

Figure 2A .Figure 2B .Figure 3A .Figure 3B .
Figure2A.Oestradiol secretion by porcine granulosa cells derived from small (1-2 mm) follicles assayed by RIA after 48 h of in vitro culture in the presence of ICI 164.384 (10 -7 M) and cyclofenil (10 -7 M) applied individually and in combination in basic conditions and in FSH (50 ng/ml) stimulated conditions.Each bar represents mean ± SEM for 5 experiments performed in triplicates.** denotes means significantly different from control (Ctrl for basic conditions, FSH for FSH-stimulated conditions; P<0.01)

Figure 4A .Figure 4B .
Figure4A.Telomerase activity in porcine granulosa cells derived from small (1-2 mm) follicles assayed using TRAPEZE Telomerase® Detection Kit after 48 h of in vitro culture in the presence of ICI 164.384 (10 -7 M) and cyclofenil (10 -7 M) applied individually and in combination in basic conditions and in FSH (50 ng/ml) stimulated conditions.Each bar represents mean ± SEM for 5 experiments performed in triplicates.** denotes means significantly different from control (Ctrl for basic conditions, FSH for FSH-stimulated conditions; P<0.01)