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Reproduction (2006) 131 545-553
DOI: 10.1530/rep.1.00885
Copyright © 2006 Society for Reproduction and Fertility
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RESEARCH

Hormonal regulation of expression of growth differentiation factor-9 receptor type I and II genes in the bovine ovarian follicle

Barana C Jayawardana, Takashi Shimizu, Hiromi Nishimoto1, Etsushi Kaneko, Masafumi Tetsuka1 and Akio Miyamoto

Graduate School of Animal and Food Hygiene and 1 Department of Agriculture and Life Science, Obihiro University of Agriculture and Veterinary Medicine Inada-Machi, Obihiro, Hokkaido, Japan 080-8555

Correspondence should be addressed to T Shimizu; Email: shimizut{at}obihiro.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Growth differentiation factor-9 (GDF-9) and bone morphogenetic proteins (BMPs) are crucial factors in follicular growth and development. GDF-9 and BMPs initiate signaling by assembling type I (ALK-3, ALK-5 and ALK-6) and type II (BMPRII) receptors. However, the mechanism regulating the expression of these receptors in the process of bovine follicle development is still unknown. The aim of the present study was to clarify the involvement of receptor systems for GDF-9 and BMPs in follicular selection by examining the effects of FSH and estradiol-17ß (E2) on the regulation of BMPRII, ALK-3, ALK-5 and ALK-6 mRNA expression in bovine granulosa cells (GCs). To observe mRNA expression during follicular development, follicles were obtained from heifers and classified into two groups: pre-selection follicles (PRFs) (an average of 7.7 mm follicles with low E2) and post-selection follicles (POFs) (an average of 15 mm follicles with high E2). Theca layer cells (TCs) and GCs were harvested from aspirated follicles. For in vitro studies, GCs were obtained from bovine follicles of 4–7 mm diameter and cultured in Dulbecco’s modified Eagle’s/F12 (DMEM/F-12) medium with 10% fetal calf serum for 24 h. The medium was then replaced with serum-free DMEM/F-12 supplemented with different doses of E2 (1, 10, 100 ng/ml) or FSH (1, 5, 10 ng/ml) or combinations of 1 ng/ml of E2 with different FSH doses. Total RNA was extracted and the mRNA expression of BMPRII, ALK-3, ALK-5 and ALK-6 was estimated by the quantitative real-time PCR method using a LightCycler. BMPRII and ALK-5 expression was significantly higher in the GCs of POFs than in those of PRFs, whereas ALK-3 expression was significantly lower in the GCs of POFs than in those of PRFs. There was no difference in ALK-6 expression in GCs between PRFs and POFs. The expression of BMPRII, ALK-5, ALK-3 and ALK-6 genes in the TCs was not significantly different between PRFs and POFs. Treatment of GCs with E2 alone increased BMPRII mRNA expression at a concentration of 100 ng/ml and ALK-5 mRNA expression at 10 ng/ml. BMPRII and ALK-5 mRNA levels were up-regulated by the combination of E2 (1 ng/ml) and FSH (5 ng/ml). On the other hand, FSH alone down-regulated the expression of BMPRII and ALK-5 in GCs. The results of the present study provide the first evidence that FSH and E2 regulate the expression of BMPRII and ALK-5 genes in bovine GCs. Thus, our data suggest that the GDF-9/BMPRII/ALK-5 system may be critically involved in the process of selection of bovine follicles.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Growth differentiation factor (GDF-9) and bone morphogenetic proteins (BMPs), which belong to the transforming growth factor beta superfamily, are associated with follicular development in the mammalian ovary. GDF-9 expression has been found in oocytes from rodents (McPherron & Lee 1993, McGrath et al. 1995, Hayashi et al. 1999), in bovine/ovine (Bodensteiner et al. 1999) and human (Aaltonen et al. 1999) follicles, and in porcine ovaries (Shimizu et al. 2004b). Mice deficient in GDF-9 show an arrest of follicular development beyond the primary stage (Dong et al. 1996). Direct injection of the GDF-9 gene into the pig ovary promoted follicular development (Shimizu et al. 2004a). Treatment with GDF-9 enhances primary and preantral follicular growth in vitro and in vivo (Hayashi et al. 1999, Vitt et al. 2000a) and promotes granulosa cell (GC) proliferation, modifies GC differentiation, and inhibits the steroidogenesis and luteinizing hormone (LH) receptor formation induced by follicle-stimulating hormone (FSH) (Vitt et al. 2000b). BMP-2 has been found in rat GCs and theca-interstitial cells (Erickson & Shimasaki 2003), whereas BMP-4 is expressed by theca cells (TCs) in the rat (Shimasaki et al. 1999) and bovine (Glister et al. 2004) and pig ovaries (Shimizu et al. 2004b).

GDF-9 and BMPs transmit their signals through specific receptors in the GC membrane. BMPs can bind to type II receptors (BMPRII), activin receptor (ActRIIA or ActRIIB) and type I receptors (ALK-3, ALK-5 and ALK-6). BMP-2, BMP-4 and GDF-9 have been found to interact with the type II receptor BMPRII (Liu et al. 1995, Nohno et al. 1995, Rosenzweig et al. 1995, Vitt et al. 2002). BMP-2 and BMP-4 interact with both ALK3 and ALK6 type I receptors (ten Dijke et al. 1994, Aoki et al. 2001) whereas GDF-9 interacts with ALK-5 as type I receptor (Mazerbourg et al. 2004). Although ALK-3, ALK-6 and BMPRII receptors have been identified in bovine GCs and TCs (Glister et al. 2004) as well as goat GCs (Silva et al. 2005), the presence of ALK-5 has not yet been demonstrated in ruminant follicles. Upon binding of the BMP ligand, the type II receptor trans-phosphorylates the type I receptor at an intracellular juxta-membrane site termed the GS domain, which is rich in glycine and serine residues (Wrana et al. 1992, Franzen et al. 1993). The phosphorylated type I receptor, in turn, transphosphorylates a set of intracellular substrate signaling proteins called Smads (Heldin et al. 1997, Attisano & Wrana 2000, Miyazono 2000, ten Dijke et al. 2002). The specificity of Smad signaling is determined by the type I receptors, rather than the type II receptors (Attisano & Wrana 2002). These facts suggest that receptors for GDF-9 and BMPs may be precisely controlled in a stage- and hormone-dependent manner during follicular development in the mammalian ovary.

Although the roles of GDF-9 and BMPs during follicular development have been well analyzed, the gene expression profiles of their receptors are still unknown. The aim of the present study was to examine (i) the expression of BMPRII, ALK-3, ALK-5 and ALK-6 mRNAs in pre-selection follicles (PRFs) and post-selection follicles (POFs) and (ii) whether the expression levels of these receptors are affected by steroid hormone and gonadotropin.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Acknowledgements
 References
 
Follicle collection
Paired ovaries were obtained from 21- to 26-month-old non-parous Holstein x Japanese Black F1 cattle at a local slaughterhouse. Only ovarian pairs with a corpus luteum and apparently normal follicles were used in the present study. Follicular fluid (FF) was aspirated from selected follicles using a syringe fitted with a 20 °G needle and stored at –20 °C. Follicular walls were carefully peeled off from the ovaries and individually placed in RNAlater (Ambion, Austin, TX, USA) and frozen at –30 °C. At the laboratory, the follicular walls were cut in half and GCs were harvested by gently scraping the walls with a spatula under a dissection microscope. The GC preparations obtained by this method were essentially free from contaminating thecal tissues. The remaining follicular walls were further scraped and washed several times with PBS to remove as many GCs as possible. Surrounding stroma and theca externa were also removed from the follicular walls, and the cleaned follicular walls were used as TCs. The tissue samples were then placed in RNAlater and frozen at –30 °C.

Follicles were classified into two groups, based on the diameter (POFs >8.5 mm in diameter; PRFs 7.0–8.5 mm in diameter). Each group consisted of five follicles obtained from five different cows.

Isolation and culture of bovine GCs
Ovaries were obtained at a local slaughterhouse from cows and heifers just after slaughter. After transport to the laboratory at 30 °C, the ovaries were washed three times with pre-warmed McCoy 5A medium (Sigma Chemical Co., St Louis, MO, USA). GCs were collected from medium-size follicles (4–7 mm) by aspiration using a needle (18 G) and syringe (plastic, 10 ml) and washed in Dulbecco’s modified Eagle’s/F12 (DMEM/F12) medium (Sigma). Then, the cell suspension was centrifuged, re-suspended, and seeded at a density of 2–5 x 105 cells per well (Nunc 24-well culture plates; Nalge Nunc International, NY, USA) in 1 ml DMEM/F12 containing 10% fetal calf serum (Biowest, Rue de la Caille, Nuaille, France), gentamicin 5 µl/ml and amphotericin B 10 µl/ml (Sigma). The cells were cultured for 24 h at 37 °C in a 5% CO2 atmosphere and then the wells were washed with DMEM/F12 to remove unattached cells and remaining tissue debris. The culture medium was replaced with serum-free medium supplemented with estradiol-17ß (E2, 1–100 ng/ml; Sigma), progesterone (P4, 1-100 ng/ml; Sigma), and bovine FSH (0.1–1.0 ng/ml; USDA, Alexandria, VA, USA) at several concentrations, and the culture was continued for 6 h. Treatments were terminated by aspirating medium and rinsing cells twice with PBS (Sigma), and stored in TRIZOL reagent (Invitrogen, Life technologies) at –80 °C until used for RNA extraction. This experiment was carried out three times with triplicate determinations in each.

RNA extraction
Tissue samples obtained from follicles were homogenized in denaturing solution containing 4 M guanidinium thiocyanate (Wako Pure Chemical Industries, Ltd, Osaka, Japan), 25 mM sodium citrate, 0.5% sarkosyl (Sigma) and 0.1 M ß-mercaptoethanol (Kanto Chemical Co. Inc., Tokyo Japan). Total RNA was extracted with phenol–chloroform (Chomczynski & Sacchi 1987), further purified, and treated with DNase using a commercial kit (SV total RNA Isolation System; Promega Co., Madison, WI, USA), and then frozen at –20 °C in RNA Storage Solution (Ambion).

In the cultured samples, total RNA was extracted with TRIZOL reagent following the method provide by the manufacturer and frozen at –20 °C in RNA Storage Solution.

Reverse transcription (RT) and quantitative PCR
Single-strand cDNA was reverse transcribed from total RNA (0.5–5 µg) using a 1st Strand cDNA Synthesis Kit for RT-PCR (Roche Diagnostics Co., Indianapolis, IN, USA) and random primer. The RT conditions consisted of 10 min of annealing at 25 °C, 60 min of cDNA synthesis at 42 °C, and 5 min of inactivation at 99 °C.

Genes for BMPRII, ALK-3, ALK-5, ALK-6 and ß-actin were quantified by real-time PCR with a LightCycler (Roche Diagnostics Co., Indianapolis, IN, USA) using a commercial kit (QuantiTect SYBR Green PCR; Qiagen GmbH, Hilden, Germany). The primers were designed using Primer-3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) based on the GenBank data base (Table 1Go). The amplification program consisted of an initial 15 min activation at 95 °C followed by 40 cycles of PCR (each cycle consisting of 15 s of denaturation at 94 °C, 30 s of annealing at 58 °C and 20 s of extension at 72 °C). For quantification of the target genes, a series of standards were constructed by amplifying a fragment of DNA (~700 bp) that contained the target sequence for real-time PCR (100 ~ 150 bp). The PCR products were subjected to electrophoresis, and the target band was cut out and purified using a DNA purification kit (SUPREC-01; TaKaRa Bio. Inc., Otsu, Japan) for DNA standard. Five to eight serially diluted DNA standards were included in every PCR run. Standard curve obtained with several dilutions of representative genes (BMPRII and ALK-5) from crossing points (cycle numbers) plotted against the logarithmic concentration of the serial dilutions are shown in the Fig. 1Go. The values were normalized using ß-actin as the internal standard. ß-Actin mRNA has been found in pig follicle cells with levels that are independent of follicle status and size (Tilly et al. 1992). In addition, its expression is not affected by growth factors and gonadotropins (LaPolt et al. 1990, Weiner & Dias 1993).


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  		Table 1 Primer pairs used for detection of mRNAs.
 

Figure 1
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  		Figure 1 Standard curves obtained with several dilutions of BMPRII and ALK-5 from crossing points (cycle numbers) plotted against the logarithmic concentration of the serial dilutions.

 
Steroid hormone extraction and assay
Hormone assays were done after extraction with diethyl ether as described previously (Acosta et al. 1998). The concentrations of the different hormones were estimated in duplicate by double-antibody enzyme immunoassay (EIA) using 96-well ELISA plates (Corning Glass Works, Corning, NY, USA). The EIA for E2 and P4 was performed as previously described (Miyamoto et al. 1992, Acosta et al. 1998, Wijayagunawardane et al. 1998). Standard curves were made for 2-2000 pg/ml E2 and 0.05–50 ng/ml P4. The ED50 values of the assay for E2 and P4 were 70 pg/ml and 1.0 ng/ml respectively. The average intra- and inter-assay coefficients of variation were 6.3 and 9.5% for E2 and 6.5 and 9.7% for P4. The average post-extraction recoveries for E2 and P4 were 80 and 85% respectively.

Data analysis
All data are presented as means ± S.E.M. The differences of expression of BMPRII, ALK-3, ALK-5, and ALK-6, and of the concentrations of E2 and P4 in FF between POFs and PRFs were analyzed by the Student’s t-test. Levels of several factors in treated bovine GCs were tested for significant differences using ANOVA, followed by the Fisher’s LSD test as a multiple comparison test. Differences were considered significant at P<0.05 or less.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Acknowledgements
 References
 
Expression of BMPRII, ALK-3, ALK-5 and ALK-6 mRNAs in the PRF and POF
Table 2Go shows the characteristics of the ovarian follicles used in this study. The concentrations of E2 and P4 in FF were significantly higher in the POFs than in the PRFs. The expression of BMPRII and ALK-5 mRNAs in the GCs were significantly higher in the POFs than in the PRFs we examined (Fig. 2AGo and Fig. 3BGo). In contrast, the expression of ALK-3 mRNA was significantly lower in the GCs of POFs than in the PRFs (Fig. 3AGo). The expression of ALK-6 mRNA did not significantly differ between the PRF and the POF GCs (Fig. 3CGo). The mRNA expression of BMPRII, ALK-3, ALK-5 and ALK-6 in TCs showed no significant differences between PRFs and POFs (Fig. 2BGo, Fig. 3DGo–F).


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  		Table 2 Mean follicle diameter, the concentrations of estradiol (E2) and progesterone (P4) in follicular fluid and E2/P4 ratio in each follicle from bovine ovary.
 

Figure 2
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  		Figure 2 Expression of BMPRII mRNA in the GCs (A) and TCs (B) of pre-selection follicles (PRFs, n = 5) and post-selection follicles (POFs, n = 5). The data are means ± S.E.M. *P<0.05 significant difference. The expression of each factor is normalized on the basis of ß-actin expression.

 

Figure 3
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  		Figure 3 Expression of ALK-3, ALK-5 and ALK-6 mRNAs in the GCs (A–C) and TCs (D–F) of pre-selection follicles (PRFs, n = 5) and post-selection follicles (POFs, n = 5). The data are means ± S.E.M. *P<0.05 and ***P < 0.001 significant differences. The expression of each factor is normalized on the basis of ß-actin expression.

 
Effects of E2 and FSH on expression of BMPRII and ALK-5
Because the expression of BMPRII and ALK-5 mRNAs in POFs were high, we examined the effect of E2 on these mRNAs using bovine GCs. A high concentration of E2 (100 ng/ml) significantly increased the expression of BMPRII mRNA in GCs (Fig. 4AGo). On the other hand, the expression of ALK-5 mRNA was significantly increased when the E2 concentration was 10 ng/ml, and the increase was maintained at 100 ng/ml E2 (Fig. 4BGo). In contrast, treatment with FSH alone down-regulated the expression of the BMPRII and ALK-5 genes in cultured bovine GCs (Fig. 4C and DGo).


Figure 4
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  		Figure 4 Effects of E2 (A and B) and FSH (C and D) on expression of BMPRII and ALK-5 mRNAs in cultured bovine GCs. The indicated concentrations of E2 and FSH were added to the culture medium. The data are means ± S.E.M. of three experiments with triplicate determinations in each. Different superscripts denote significantly different values (P<0.05).

 
Effects of E2 and P4 on expression of ALK-3
Since the expression of the ALK-3 gene was lower in the GCs of the POFs than in the GCs of the PRFs, we examined the effects of E2 and P4 on ALK-3 gene expression. The expression of the ALK-3 gene in cultured GCs was not stimulated by E2 or P4 (Fig. 5A and BGo).


Figure 5
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  		Figure 5 Effects of E2 (A) and P4 (B) on expression of ALK-3 mRNA in cultured bovine GCs. The indicated concentrations of E2 and P4 were added to the culture medium. The data are means ± S.E.M. of three experiments with triplicate determinations in each.

 
Effect of combination treatment with FSH and E2 on expression of BMPRII and ALK-5
At a constant concentration of 1 ng/ml E2, the expression of BMPRII and ALK-5 mRNAs significantly increased when the FSH concentration was 5 ng/ml, and the increase was maintained at a concentration of 10 ng/ml FSH (Fig. 6A and BGo).


Figure 6
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  		Figure 6 Effects of E2 and FSH on the expression of BMPRII (A) and ALK-5 (B) in cultured bovine GCs. The indicated concentrations of E2 and FSH were added to the culture medium. The data are means ± S.E.M. of three experiments with triplicate determinations in each. Different superscripts denote significantly different values (P<0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Acknowledgements
 References
 
The present study provides the first evidence that ALK-5 is expressed in the GCs and TCs of PRFs and POFs from the bovine ovary, and that E2 and FSH enhance the expression of the BMPRII and ALK-5 genes in bovine cultured GCs. The expression of ALK-5/TßRI has been reported in hamster ovary (Roy 2000) and porcine GCs (Goddard et al. 1995), but it has not been reported in any ruminant ovary, whereas BMPRII expression in bovine GCs and TCs has been reported (Glister et al. 2004). In the present study, the expression of mRNAs for BMPRII and ALK-5 was significantly higher in the GCs of the POFs than in those of the PRFs. The fact that the expression of BMPRII, ALK-3 ALK-5 and ALK-6 in TCs was not significantly different between PRFs and POFs suggests that the expression of these receptors is not developmentally regulated in bovine TCs. BMPRII, ALK-3, ALK-5 and ALK-6 are essential for transducing the biological action of GDF-9, BMP-2 and BMP-4 (ten Dijke et al. 1994, Liu et al. 1995, Nohno et al. 1995, Rosenzweig et al. 1995, Aoki et al. 2001, Vitt et al. 2002, Mazerbourg et al. 2004). Thus, the results of the present study suggest that BMPRII and ALK-5 may be physiologically relevant factors for transducing signals critical for follicular development in cattle. Such signaling could well be the result of the action of GDF-9.

The expression of ALK-3 and ALK-6 has been reported in rats (Shimasaki et al. 1999), pigs (Quinn et al. 2004), sheep (Souza et al. 2002) and cattle (Glister et al. 2004) GCs and TCs. In the early stages of rat follicular development the levels of ALK-3 and ALK-6 mRNA expressions in the GCs increased and abundant expression was maintained in the tertiary and dominant follicular stages (Erickson & Shimasaki 2003). Therefore, the present study confirms the expression of ALK-3 and ALK-6 mRNA in GCs of dominant follicles and further showed that ALK-3 mRNA expression significantly decreased in the GCs of the POFs compared with those of the PRFs, whereas ALK-6 expression did not differ significantly. While information is currently lacking, it has not yet been confirmed that both the polyovular species rats and the monoovular species bovines have the same pattern of BMP/GDF-9 receptor expression during follicular development. In addition, further studies will be necessary to clarify the mechanism of regulation of ALK-3 mRNA expression in bovine GCs. On the other hand mRNA expression of BMPRII and ALK-5 significantly increased in POF GCs compared with PRF GCs, suggesting that ALK-5 may be playing a very important role in follicular selection by activating the specific intracellular substrate protein called Smads in response to the GDF-9 ligand binding to the BMPRII receptor during follicular development.

The FF of dominant follicles has higher E2 than that of subordinate follicles in the bovine ovary (Bodensteiner et al. 1996, Evans et al. 1997, Ginther et al. 1997). In addition, previous studies suggested that the selection of follicles in cattle occurs when the largest follicle of the cohort of growing follicles reaches an average size of 8.5 mm (Ginther et al. 2001). Our results confirmed that the E2 concentration in FF of the POFs was much higher than that in the FF of PRFs. Since the follicles used in the present study were obtained during the period when E2 was increasing in the follicular environment, we examined the effects of E2 on the expression of BMPRII, ALK-3 and ALK-5 mRNAs using cultured GCs.

Our results showed that the expression of BMPRII and ALK-5 mRNA was stimulated by E2, whereas ALK-3 expression was not changed by E2 in bovine GCs. The expression of the BMPRII and ALK-5 genes was significantly increased when the E2 concentration was 100 ng/ml and 10 ng/ml respectively. E2 is one of the major factors effecting follicle selection in monoovular species such as cattle (Ginther et al. 2001). In the present study, the E2 concentration in FF of POFs was 88.7 ± 17.8 ng/ml, so BMPRII and ALK-5 may be up-regulated during the process of follicular selection in vivo.

The more-developed largest follicle not only withstood but also required the low FSH concentrations associated with deviation. The FSH surge that stimulates the emergence of a wave begins to decline when the largest follicle is 4–5 mm in heifers, and the interval from the beginning of the FSH decline to the beginning of deviation is about 3 days. Experimental reduction of FSH below the concentrations at the middle of the FSH decline is associated with a decrease in diameter of the largest follicles (Bergfelt et al. 2000, Ginther et al. 2000). These results indicate that FSH plays a crucial role in the follicular selection/deviation during bovine follicle development. Indeed, the expression of the BMPRII and ALK-5 genes in GCs from 4–7 mm follicles in our culture increased when FSH was added to follicles treated with a constant concentration of 1 ng/ml E2. This result further suggests that BMPRII and ALK-5 may be associated with follicular selection in the bovine ovary. Interestingly, our data indicated that FSH alone down-regulates the expression of the BMPRII and ALK-5 genes, suggesting that E2 is required for expression of BMPRII and ALK-5 during follicular development.

In bovine GCs, LH receptor expression begins when the mean diameter of follicles reaches around 8 mm or above (nearly 36 h after recruitment) (Bao & Garverick 1998). For our in vitro cell culture model, we obtained GCs from bovine follicles of less than 7 mm in diameter. Therefore, we did not examine the effect of LH on the expression of BMPRII and ALKs in this culture system. However, at the late follicular development stage, especially after deviation, LH plays a key role in follicular growth, including the production of E2 (Ginther et al. 2001). Therefore, future studies need to examine the effect of LH on BMPRII and ALK-5 receptor expression in bovine GCs using a cell culture system in which GCs are obtained from follicles of 8–10 mm diameter.

In conclusion, the findings of the present study provide strong evidence that FSH and E2 cooperatively play physiological roles in regulating GDF-9 type I and type II receptor expression in the GCs during bovine follicular development. Thus, it is proposed that the GDF-9/BMPRII/ALK-5 system may be crucially involved in the process of selection of bovine follicles.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
References
 
The authors thank Dr K Okuda, Okayama University Japan, for progesterone antibodies. This study was supported by the 21st Century COE program (A-1), Ministry of Education, Culture, Science and Technology, Japan, a Grant-in-Aid for Scientific Research of the Japan Society for the Promotion of Science (JSPS) and the Akiyama Foundation, Japan. E K is a postdoctoral fellow supported by the COE program. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.


   Footnotes
 
Received 14 July 2005
First decision 20 September 2005
Revised manuscript received 22 November 2005
Accepted 5 December 2005


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 

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