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Apelin and APJ receptor expression in granulosa and theca cells during different stages of follicular development in the bovine ovary: Involvement of apoptosis and hormonal regulation
Animal Reproduction Science 116 (2009) 28–37
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Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci
Apelin and APJ receptor expression in granulosa and theca cells during different stages of follicular development in the bovine ovary: Involvement of apoptosis and hormonal regulation Takashi Shimizu a,∗, Naomichi Kosaka a, Chiaki Murayama a, Masa Tetsuka b, Akio Miyamoto a a b
Graduate School of Animal and Food Hygiene, Obihiro University of Agriculture and Veterinary Medicine, Obihiro 080-8555, Japan Department of Agriculture and Life Science, Obihiro University of Agriculture and Veterinary Medicine, Obihiro 080-8555, Japan
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Article history: Received 15 September 2008 Received in revised form 24 December 2008 Accepted 19 January 2009 Available online 24 January 2009 Keywords: Apelin APJ receptor Follicle Progesterone Apoptosis Granulosa cell Theca cell
a b s t r a c t In the mammalian ovary, the microvasculature in the thecal layer of follicles is associated with follicular development. Apelin and its receptor, APJ, are expressed in the tissues and organs which include the vasculature. The aims of the present study were to examine the mRNA expression of apelin and the APJ receptor in granulosa cells and theca tissue of bovine follicles and the effects of steroid hormone and gonadotrophins on the expression of these genes in cultured granulosa cells and theca cells. The expression of apelin mRNA was not found in the granulosa cells of bovine follicles. The expression of the APJ gene was increased in granulosa cells of estrogen-inactive dominant follicles. The expression of apelin mRNA increased in theca tissues of estrogen-inactive dominant follicles. APJ expression in theca tissues increased with follicle growth. Progesterone stimulated the expression of APJ mRNA in the cultured granulosa cells. FSH stimulated the expression of APJ mRNA in the cultured granulosa cells. LH induced the expression of apelin and APJ receptor mRNAs in cultured theca cells. Taken together, our data indicate that the APJ receptor in granulosa cells and both apelin and the APJ receptor in theca tissues are expressed in bovine ovary, that APJ in granulosa cells may be involved in the appearance of the cell apoptosis, and that LH stimulates the expression of apelin and APJ genes in theca cells. © 2009 Elsevier B.V. All rights reserved.
∗ Corresponding author. Fax: +81 155 49 5419. E-mail address: [email protected] (T. Shimizu). 0378-4320/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2009.01.009
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1. Introduction During the follicular phase of the estrous cycle in the mammalian ovary, one follicle is selected to mature to the preovulatory stage while all other follicles undergo atresia. It has been suggested by several studies that vasculature may play an important role in this process (Zeleznik et al., 1981; Jiang et al., 2004). Angiogenic factors are associated with follicular vasculature during follicular development (Shimizu et al., 2002). In fact, direct ovarian injection of the vascular endothelial growth factor (VEGF) gene that induces vascular formation produced a large number of preovulatory follicles as the vasculature grew in pigs (Shimizu et al., 2003) and rats (Shimizu et al., 2007, 2008). Thus, ovarian follicular development is affected by the vasculature that is established around the follicle. Apelin was identified as an endogenous ligand of the orphan G-protein-coupled receptor APJ from bovine stomach extracts (Tatemoto et al., 1998). Rat, mouse, cow, and human apelin cDNAs have been characterized (Edinger et al., 1998; Tatemoto et al., 1998). These cDNAs encode a 77-amino-acid preproprotein, and a 36-amino-acid variant of apelin appears to be the parent peptide. In all species cloned to date, a sequence of 23 amino acids in the C-terminal region is conserved, implying that the C-terminal region is critical for biological activity. APJ is a 380 amino acid seven-transmembrane domain Gi-coupled receptor that is most closely related to the angiotensin II receptor (O’Dowd et al., 1993). A previous study reported that the apelin–APJ system is associated with vessel formation in the retina. During the formation of retinal vessels, APJ mRNA is associated with vessels as they form and traces the centrifugal extension of the superficial vasculature, while apelin mRNA expression is up-regulated at the leading edge of vessel formation (Saint-Geniez et al., 2002). In addition, possible functional roles for apelin/APJ signals in endothelial cells were reported. Vasodilatory effects are one of such possible action of apelin (Tatemoto et al., 2001; Ishida et al., 2004). The purpose of the present study was to examine the localization and expression of apelin and the APJ receptor in follicles at different developmental stages in the bovine ovary, which has a large number of vessels. In addition, we examined whether the expression levels of apelin and APJ receptor in granulosa and theca cells are affected by steroid or gonadotropin hormones. 2. Materials and methods 2.1. Materials Dulbecco’s modified Eagle’s /F12 (DMEM/F12), amphotericin B, gentamicin, estradiol-17 (E2), progesterone (P4), phosphate-buffered saline (PBS), and sarkosyl were purchased from Sigma Chemical Co., St. Louis, MO, USA. Fetal calf serum (FCS) was obtained from Biowest, Rue de la Caille, Muaille, France. Antiserum for apelin and APJ was purchased from Phoenix Pharmaceuticals, Inc., Belmont, CA, USA. 2.2. Collection of follicles and isolation of granulosa and theca cells Ovaries were obtained from Holstein × Japanese Black F1 heifers at a local slaughterhouse. Each ovarian pair was placed in a fabric paper bag, frozen at −20 ◦ C and transported to the laboratory. At the laboratory, ovaries were examined macroscopically, and the number and status of follicles and corpus luteum (CL) were recorded. The diameters of follicles were determined from the weight of the follicular fluid as previously described by Murasawa et al. (2005). CLs were macroscopically assessed for color, vascularity, and consistency using published criteria (Ireland et al., 1980) and classified into four stages (stages I–III: luteal phase, and stage IV: follicular phase). Follicles were classified into four categories using calculated follicle diameters, concentrations of E2 and the ratio of E2 to progesterone (E/P) in the follicular fluid. The categories were as follows: small follicles (mean diameter: 7.4 mm, SF), estrogen-inactive dominant follicles (mean diameter: 13.5 mm, E/P < 1: EID with stages I–III CL), estrogen active dominant follicles (mean diameter: 13.5 mm, E/P ≥ 1: EAD with stages I–III CL) and preovulatory follicles (mean diameter: 16.8 mm, E/P ≥ 1: POF with stage IV CL). Each group consists of five follicles obtained from five different cows. Follicular walls were carefully peeled off from the ovaries and individually placed in the RNA later and frozen at −30 ◦ C.
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The follicular walls were cut 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 much GCs as possible. Surrounding stroma and theca externa were also removed from the follicular walls, and the cleaned follicular walls were used as TCs. 2.3. Granulosa cell culture Ovaries were obtained from cows and heifers (Holstein breed) at a local slaughterhouse. Granulosa cells were collected from follicles (5–6 mm) by aspiration and filtrated through a stainless steel filter (45 m, TOKYO SCREEN CO., LTD., Tokyo, Japan) to remove oocytes. Then, the cells were centrifuged at 800 × g and washed twice in culture medium; DMEM/F12 medium containing amphotericin B (10 l/ml) and gentamicin (5 l/ml). The cells were cultured in culture medium containing 10% FCS for 24 h at 37 ◦ C in 5% CO2 and then the wells were washed with PBS to remove unattached cells. To examine the expression of APJ on granulosa cell apoptosis, the culture medium was replaced with serum-free medium supplemented with or without 100 ng/ml FSH and the cultures were continued for 48 h. To examine the effect of steroids on the expression of the APJ gene, the culture medium was replaced with serum-free medium supplemented with oestradiol-17 (1 ng/ml or 10 ng/ml; Sigma Chemical Co., St. Louis, MO, USA), progesterone (P4, 1–100 ng/ml; Sigma Chemical Co., St. Louis, MO, USA) at various concentrations, and the culture was continued for 6 h. The treatments were terminated by aspirating medium and rinsing the cells twice with phosphate-buffered saline (Sigma Chemical Co., St. Louis, MO, USA), and were then stored in TRIZOL® at −80 ◦ C until used for RNA extraction. The replication for culture experiment performed three experiments with triplicate in each. 2.4. Hoechst 33342 staining Hoechst staining was performed to confirm the apoptotic profile as a result of morphological changes in the nucleus in which Hoechst 33342 binds specifically to the A–T base region in DNA and emits fluorescence. The cultured cells were rinsed in PBS and fixed with 1% glutaraldehyde (Wako) in PBS at 4 ◦ C for 24 h. Then the cells were stained with Hoechst 33342 (50 g/l) in PBS for 3 min. The proportion of cells with nuclear fragmentation was calculated by counting the number of stained cells per 200 cells. 2.5. Theca cell culture Ovaries were obtained from cows and heifers (Holstein breed) at a local slaughterhouse. Theca cells were isolated from the ovaries using the following method (Murayama et al., 2008). Briefly, large follicles (>10 mm in diameter) were selected and follicular fluid was aspirated using a syringe with a 22-gauge needle. The follicles were opened by making a small incision on the surface. Granulosa cells were removed by gentle scraping with a medicine spoon under a stereomicroscope. We checked the complete removal of granulosa cells under a stereomicroscope. The thecal layer was placed into PBS containing 2 mg/ml collagenase (452 U/mg, type 1, Sigma), 1 mg/ml hyaluronidase (391 U/mg, type VIII, Sigma), 1 mg/ml protease (4.5 U/mg, Sigma), and 0.4% (v/v) bovine serum albumin, and the dissociation reaction was performed for 40 min at 37 ◦ C. Centrifugal separation was carried out by 350 × g. Then, Tris–HCl buffer (pH 8.0) was put into the tube for 1 min at 37 ◦ C. The dispersed cells were washed twice with PBS. Theca cells were seeded at a density of 1 × 105 cells per well (24-well culture plate) in 1 ml of Dulbecco’s modified Eagle’s /F12 medium (DMEM/F12; Sigma Chemical Co.) containing amphotericin B (10 l/ml), gentamicin (5 l/ml), and 5% fetal calf serum (FCS; Biowest, Rue de la Caille, Muaille, France) as a pre-incubation for 24 h. After pre-incubation, the culture medium was replaced with serum-free medium supplemented with or without 100 ng/ml LH, and the cultures were continued for 24 h and 48 h. The treatments were terminated by aspirating medium and by rinsing the cells
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twice with phosphate-buffered saline, and were then stored in TRIZOL® at −80 ◦ C until used for RNA extraction. 2.6. RNA extraction and reverse transcriptase (RT) Follicle tissue samples were homogenized in denaturing solution containing 4 M guanidium thiocyanate (Wako Pure Chemical Industries Ltd., Osaka, Japan), 25 mM sodium citrate (Wako), 0.5% sarkosyl (Sigma), and 0.1 M beta-mercapto ethanol (Kanto Chemical Co. Inc., Tokyo, Japan). Total RNA was extracted with phenol–chloroform, purified further, 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 Inc., TX, USA). In the cultured samples total RNA was extracted with TRIZOL® regent (Invitrogen Japan K.K.) following the method provide by the manufacturer and frozen at −20◦ C in RNA storage solution. Single-strand cDNA was reverse transcribed from total RNA (500 ng) using a 1st Strand cDNA Synthesis Kit for RT-PCR (Roche Diagnostics Co., Indianapolis, IN, USA) and random primer. 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. 2.7. Quantitative PCR Abundances of mRNA for apelin and the APJ receptor were quantified by real-time PCR with iQ5Cycler (Bio-Rad Laboratories, CA, USA) using a commercial kit (QuantiTectTM SYBR® Green PCR: QIAGEN GmbH, Hilden, Germany). The primers used for an amplification of the apelin, APJ receptor, and -actin mRNA were as follows: apelin (106 bp) forward: 5 -aag gca cca tcc gat acc tg-3 , reverse: 5 -atg gga ccc ttg tgg gag a-3 ; APJ receptor (100 bp) forward: 5 -tct ggg cca cct aca cct at-3 , reverse: 5 -acg ctg gcg tac atg ttg-3 ; -actin (256 bp) forward: 5 -cca agg cca acc gtg aga aga t-3 , reverse: 5 -cca cgt tcc gtg agg atc ttc a-3 . The amplification program consisted of 15 min for activation at 95 ◦ C followed by 40 cycles of PCR (94 ◦ C for 15 s, specific annealing temperature for each factor for 30 s and 72 ◦ C for 20 s). 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. The PCR products were subjected to electrophoresis, and the target band was cut out and purified using a DNA purification kit (SUPRECTM -01, TaKaRa Bio. Inc., Otsu, Japan) for DNA standard. Five to seven serially diluted DNA standards were included in every PCR run. Standard curve was obtained with several dilutions of target genes (apelin and APJ) from crossing points (cycle numbers) plotted against the logarithmic concentration of the serial dilutions. The values were normalized using -actin as the internal standard. -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 and Dias, 1993). 2.8. Data analysis All data are presented as mean ± S.E.M. The level of several factors in the follicles at different developmental stages and the treated bovine granulosa cells or theca cells was tested for significant differences using ANOVA, followed by the Tukey–Kramer test as a multiple comparison test. The differences of expression of the APJ receptor in cultured granulosa cells with or without FSH were analyzed by the Student’s t-test. Differences were considered significant at p < 0.05 or less. 3. Results 3.1. Expression of apelin and APJ mRNA in granulosa cells The expression of apelin mRNA was not found in the granulosa cells of follicles at different developmental stages (Fig. 1A). On the other hand, expression of the APJ gene was significantly
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Fig. 1. (A) Representative electrophoresis image and (B) quantitative expression of APJ receptor mRNA in granulosa cells of small (SF, n = 5), estradiol inactive dominant (EID, n = 5), estradiol active dominant (EAD, n = 5), and preovulatory follicles (POF, n = 5). Each group consists of five follicles obtained from five different cows. Different superscripts denote significantly different values (p < 0.05). M shows a molecular marker (100 bp ladder).
increased in granulosa cells of estrogen-inactive dominant follicles compared with others (Fig. 1A and B). 3.2. The effect of progesterone and estradiol on the expression of APJ in granulosa cells We found a high expression of APJ mRNA in the estrogen-inactive dominant follicles compared with other follicles. The ratio of E2/P4 of EID follicles is lower level than one. Therefore, we examined the effect of progesterone alone and the combination of progesterone and estradiol on the expression of APJ mRNA in the granulosa cells. The level of APJ mRNA in cells treated with 1 ng/ml progesterone plus 10 ng/ml estradiol, or 10 ng/ml progesterone plus 1 ng/ml estradiol was increased compared with the levels in those treated with 1 ng/ml or 10 ng/ml progesterone alone (Fig. 2A and B). 3.3. Granulosa cell apoptosis and APJ expression Since APJ receptor expression is high in estrogen-inactive dominant follicles which undergo atresia (granulosa cell apoptosis), we examined the relationship between granulosa cell apoptosis and APJ expression in cultured granulosa cells. The incidence of granulosa cell apoptosis was determined by Hoechst 33342 nuclear staining in granulosa cells. FSH suppressed the granulosa cell apoptosis when the cells are cultivated in serum-free medium (Fig. 3A), and inhibited the expression of APJ receptor in the granulosa cells at 48 h of vitro culture (Fig. 3B). 3.4. Expression of apelin and APJ mRNA in theca tissues The expression of apelin mRNA significantly increased in theca tissues of estrogen-inactive dominant follicles compared with others (Fig. 4A and B). Except estrogen-inactive dominant follicles, APJ expression increased with follicle growth (Fig. 4C).
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Fig. 2. Effects of progesterone (A) and the combination of progesterone and estradiol (B) on the expression of APJ receptor mRNA in cultured granulosa cells. The data are shown as mean ± S.E.M. of three experiments with triplicate determination in each. Different superscripts denote significantly different values.
3.5. The effect of LH on APJ and apelin expression in theca cells Since a large number of factors derived in theca cells are influenced by LH, we examined the effect of LH on the expression of apelin and APJ receptor mRNAs in theca tissues. LH stimulated the expression of apelin and APJ receptor mRNAs at 24 h and 48 h of culture period, respectively (Fig. 5A and B). 4. Discussion In the mammalian ovary, the follicle acquires a vascular wreath in the theca layer which, when fully established, consists of two concentric networks of vessels in the theca interna and externa, respectively (Reynolds et al., 1992; Findlay, 1986; Gordon et al., 1995; Augustin et al., 1995; Goede et al., 1998). The establishment of the vascular wreath, particularly the expansion of the inner capillary plexus of the theca interna, coincides with a period of rapid growth and differentiation of the follicle. The differences in microvasculature in the thecal layer of follicles are an important underlying cause of follicular heterogeneity such as dominance or atresia. Such vascular formation in the theca layer during follicular development is regulated by angiogenic factors (Shimizu et al., 2003). Recently, it has been reported that both apelin and the APJ receptor are expressed in the vascular system (Devic et al., 1999; Kleinz and Davenport, 2004). Our present study indicated that the APJ receptor is expressed in granulosa cells, and that both apelin and the APJ receptor are expressed in the theca cell layer of follicles in bovine ovaries. In addition, we found that the expression of APJ receptor mRNA increased in theca tissue of POF, in which a large amount of blood vessels exist in the theca tissue, compared with those of SF. However, apelin expression in granulosa cells decreased in POF and EAD follicles
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Fig. 3. FSH suppresses granulosa cell apoptosis. (A) The granulosa cells were cultured with 100 ng/ml of FSH for 24 h and 48 h. (B) FSH suppressed the expression of APJ receptor in culture granulosa cells for 48 h. Each data point represents the mean ± S.E.M. of three experiments with triplicate determinations. An asterisk denotes a significant difference at p < 0.05.
than in EID follicles. Therefore, our findings suggested that the apelin/APJ system may not strongly be associated with thecal vasculature during follicular development in the bovine ovary. In this study, APJ receptor mRNA, but not apelin mRNA, was expressed in granulosa cell of follicles at different developmental stages. Although the EAD and EID follicles that were used in the present study had the same diameter, the APJ receptor mRNA expression in the granulosa cells of EID follicles was higher than that in the EAD follicles. The EID follicles in the present study represent the period of decreasing estradiol and increasing progesterone in the environment. Such a follicle undergoes atresia, which is induced by granulosa cell apoptosis (Tilly et al., 1991; Hsueh et al., 1994; Liu et al., 2003). We examined the effect of progesterone and estradiol on the expression of APJ receptor mRNA in granulosa cells. The expression of APJ receptor mRNA is induced by progesterone, which is present in high levels in the follicular fluid of atretic follicles, and was not suppressed when estradiol was added to the culture medium. Moreover, we examined the involvement of AJP receptor expression and granulosa cell apoptosis using an in vitro culture system. The expression of APJ mRNA significantly increased when granulosa cell apoptosis was induced. These data suggest that the APJ receptor may be associated with the follicular atresia induced by granulosa cell apoptosis during bovine follicular development. Therefore, the results from this study indicate that the increase in expression of APJ receptor mRNA in granulosa cells may be one of the characteristics of follicular atresia. We found that mRNA of both apelin and the APJ receptor are expressed in the theca cell layer of bovine follicles. Interestingly, mRNA expression of apelin is increased in the theca cell layer of EID follicles compared with those of others. In addition, we observed that the expression of APJ receptor
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Fig. 4. Expression of (A) apelin and (B) APJ receptor mRNA in theca tissues of small (SF, n = 5), estradiol inactive dominant (EID, n = 5), estradiol active dominant (EAD, n = 5), and preovulatory follicles (POF, n = 5). Each group consists of five follicles obtained from five different cows. Different superscripts denote significantly different values (p < 0.05).
mRNA was high in the granulosa cells of EID follicle. Therefore, theca cell-derived apelin may be involved in atresia by inducing granulosa cell apoptosis. In a future study we plan to examine the effect of apelin on granulosa cell apoptosis. In this study, although the expression of APJ receptor mRNA in theca tissues increased as follicle grew, apelin expression in those tissues of the follicles except EID follicles was the constant level. Thus, these results suggested that the increase in the expression of APJ receptor mRNA in theca tissues may be associated with bovine follicular development, and that the apelin–APJ system may influence the function of theca cell by autocrine manner. We found that FSH suppressed the expression of APJ receptor mRNA in cultured granulosa cells. In the present study the expression of APJ receptor mRNA was low in the granulosa cells of SF, EAD, and POF compared with EID follicles. Therefore, FSH may contribute to the maintenance of healthy follicles by inhibiting APJ receptor during follicular development. To examine the effect of gonadotropin on the expression of apelin and APJ receptor in theca cells, we evaluated the effect of LH on the expression of apelin and APJ receptor mRNAs by using cultured theca cells. LH induced the expression of apelin and the APJ receptor in theca cells at 24 h and 48 h of culture, respectively. Although the expression pathway for apelin and APJ by LH is still unknown, the different expression timing on those factors may depend on the transcription pathway. LH is essential to stimulate antral follicles to grow beyond 9 mm in diameter when a shift from FSH- to LH-dependency has taken place (Gong et al., 1996). The mean diameter of EAD and POF follicles in the present study was 13.5 mm and 16.8 mm, respectively, and these follicles have a high sensitivity to LH. Therefore, our results suggested that the apelin/APJ system in theca tissue may play an important role for in follicle growth to the ovulatory phase. In conclusion, the present study provides the first evidence that apelin in granulosa cells, and apelin and APJ in theca tissue are expressed in bovine follicles. Our findings demonstrate that the APJ receptor in granulosa cells may be associated with follicular atresia, and that aplein and APJ expression in the
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Fig. 5. Effect of LH on the expression of (A) apelin and (B) APJ receptor mRNAs in cultured theca cells. The data are shown as mean ± S.E.M. of three experiments with triplicate determination in each. An asterisk denotes a significant difference at p < 0.05.
theca cell is induced by LH. Thus, the apelin/APJ system may contribute to the function of granulosa and theca cells rather than the formation of follicular vasculature. References Augustin, H.G., Braun, K., Telemenakis, I., Modlich, U., Kuhn, W., 1995. Ovarian angiogenesis: phenotypic characterization of endothelial cells in a physiological model of blood vessel growth and regression. Am. J. Pathol. 147, 339–351. Devic, E., Rizzoti, K., Bodin, S., Knibiehler, B., Audigier, T., 1999. Amino acid sequence and embryonic expression of msr/apj. Mech. Develop. 84, 199–203. Edinger, A.L., Hoffman, T.L., Sharron, M., Lee, B., Yi, Y., Choe, W., Kolson, D.L., Mitrovic, B., Zhou, Y., Faulds, D., Collman, R.G., Hesselgesser, J., Horuk, R., Doms, R.W., 1998. An orphan seven-transmembrane domain receptor expressed widely in the brain functions as a coreceptor for human immunodeficiency virus type 1 and simian immunodeficiency virus. J. Virol. 72, 7934–7940. Findlay, J.K., 1986. Angiogenesis in reproductive tissues. J. Endocrinol. 111, 357–366. Goede, V., Schmidt, T., Kimmina, S., Kozian, D., Augustin, H.G., 1998. Analysis of blood vessel maturation processes during cyclic ovarian angiogenesis. Lab. Invest. 78, 1385–1394. Gong, J.G., Campbell, B.K., Bramley, T.A., Gutierrez, C.G., Peters, A.R., Webb, R., 1996. Suppression in the secretion of folliclestimulating hormone and luteinizing hormone, and ovarian follicle development in heifers continuously infused with a gonadotropin-releasing hormone agonist. Biol. Reprod. 55, 68–74. Gordon, J.D., Shifren, J.L., Foulk, R.A., Taylor, R.N., Jaffe, R.B., 1995. Angiogenesis in the human female reproductive tract. Obstet. Gynecol. Surv. 50, 688–697. Hsueh, A.J.W., Billig, H., Tsafriri, A., 1994. Ovarian follicle atresia: a hormonally controlled apoptotic process. Endocr. Rev. 15, 707–724. Ireland, J.J., Murphee, R.L., Coulson, P.B., 1980. Accuracy of predicting stages of bovine estrous cycle by gross appearance of the corpus luteum. J. Dairy Sci. 63, 155–160. Ishida, J., Hoshimoto, T., Hoshimoto, Y., Nishiwaki, S., Iguchi, T., Harada, S., Sugaya, T., Matsuzaki, H., Yamamoto, R., Shiota, N., Okunishi, H., Kihara, M., Uemura, S., Sugiyama, F., Yagami, K., Kasuya, Y., Mochizuki, N., Fukamizu, A., 2004. Regulatory roles for APJ, a seven-transmembrane receptor related to angiotensin-type 1 receptor in blood pressure in vivo. J. Biol. Chem. 279, 26274–26279.
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Apelin and APJ receptor expression in granulosa and theca cells during different stages of follicular development in the bovine ovary: Involvement of apoptosis and hormonal regulation Takashi Shimizu a,∗, Naomichi Kosaka a, Chiaki Murayama a, Masa Tetsuka b, Akio Miyamoto a a b
Graduate School of Animal and Food Hygiene, Obihiro University of Agriculture and Veterinary Medicine, Obihiro 080-8555, Japan Department of Agriculture and Life Science, Obihiro University of Agriculture and Veterinary Medicine, Obihiro 080-8555, Japan
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Article history: Received 15 September 2008 Received in revised form 24 December 2008 Accepted 19 January 2009 Available online 24 January 2009 Keywords: Apelin APJ receptor Follicle Progesterone Apoptosis Granulosa cell Theca cell
a b s t r a c t In the mammalian ovary, the microvasculature in the thecal layer of follicles is associated with follicular development. Apelin and its receptor, APJ, are expressed in the tissues and organs which include the vasculature. The aims of the present study were to examine the mRNA expression of apelin and the APJ receptor in granulosa cells and theca tissue of bovine follicles and the effects of steroid hormone and gonadotrophins on the expression of these genes in cultured granulosa cells and theca cells. The expression of apelin mRNA was not found in the granulosa cells of bovine follicles. The expression of the APJ gene was increased in granulosa cells of estrogen-inactive dominant follicles. The expression of apelin mRNA increased in theca tissues of estrogen-inactive dominant follicles. APJ expression in theca tissues increased with follicle growth. Progesterone stimulated the expression of APJ mRNA in the cultured granulosa cells. FSH stimulated the expression of APJ mRNA in the cultured granulosa cells. LH induced the expression of apelin and APJ receptor mRNAs in cultured theca cells. Taken together, our data indicate that the APJ receptor in granulosa cells and both apelin and the APJ receptor in theca tissues are expressed in bovine ovary, that APJ in granulosa cells may be involved in the appearance of the cell apoptosis, and that LH stimulates the expression of apelin and APJ genes in theca cells. © 2009 Elsevier B.V. All rights reserved.
∗ Corresponding author. Fax: +81 155 49 5419. E-mail address: [email protected] (T. Shimizu). 0378-4320/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2009.01.009
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1. Introduction During the follicular phase of the estrous cycle in the mammalian ovary, one follicle is selected to mature to the preovulatory stage while all other follicles undergo atresia. It has been suggested by several studies that vasculature may play an important role in this process (Zeleznik et al., 1981; Jiang et al., 2004). Angiogenic factors are associated with follicular vasculature during follicular development (Shimizu et al., 2002). In fact, direct ovarian injection of the vascular endothelial growth factor (VEGF) gene that induces vascular formation produced a large number of preovulatory follicles as the vasculature grew in pigs (Shimizu et al., 2003) and rats (Shimizu et al., 2007, 2008). Thus, ovarian follicular development is affected by the vasculature that is established around the follicle. Apelin was identified as an endogenous ligand of the orphan G-protein-coupled receptor APJ from bovine stomach extracts (Tatemoto et al., 1998). Rat, mouse, cow, and human apelin cDNAs have been characterized (Edinger et al., 1998; Tatemoto et al., 1998). These cDNAs encode a 77-amino-acid preproprotein, and a 36-amino-acid variant of apelin appears to be the parent peptide. In all species cloned to date, a sequence of 23 amino acids in the C-terminal region is conserved, implying that the C-terminal region is critical for biological activity. APJ is a 380 amino acid seven-transmembrane domain Gi-coupled receptor that is most closely related to the angiotensin II receptor (O’Dowd et al., 1993). A previous study reported that the apelin–APJ system is associated with vessel formation in the retina. During the formation of retinal vessels, APJ mRNA is associated with vessels as they form and traces the centrifugal extension of the superficial vasculature, while apelin mRNA expression is up-regulated at the leading edge of vessel formation (Saint-Geniez et al., 2002). In addition, possible functional roles for apelin/APJ signals in endothelial cells were reported. Vasodilatory effects are one of such possible action of apelin (Tatemoto et al., 2001; Ishida et al., 2004). The purpose of the present study was to examine the localization and expression of apelin and the APJ receptor in follicles at different developmental stages in the bovine ovary, which has a large number of vessels. In addition, we examined whether the expression levels of apelin and APJ receptor in granulosa and theca cells are affected by steroid or gonadotropin hormones. 2. Materials and methods 2.1. Materials Dulbecco’s modified Eagle’s /F12 (DMEM/F12), amphotericin B, gentamicin, estradiol-17 (E2), progesterone (P4), phosphate-buffered saline (PBS), and sarkosyl were purchased from Sigma Chemical Co., St. Louis, MO, USA. Fetal calf serum (FCS) was obtained from Biowest, Rue de la Caille, Muaille, France. Antiserum for apelin and APJ was purchased from Phoenix Pharmaceuticals, Inc., Belmont, CA, USA. 2.2. Collection of follicles and isolation of granulosa and theca cells Ovaries were obtained from Holstein × Japanese Black F1 heifers at a local slaughterhouse. Each ovarian pair was placed in a fabric paper bag, frozen at −20 ◦ C and transported to the laboratory. At the laboratory, ovaries were examined macroscopically, and the number and status of follicles and corpus luteum (CL) were recorded. The diameters of follicles were determined from the weight of the follicular fluid as previously described by Murasawa et al. (2005). CLs were macroscopically assessed for color, vascularity, and consistency using published criteria (Ireland et al., 1980) and classified into four stages (stages I–III: luteal phase, and stage IV: follicular phase). Follicles were classified into four categories using calculated follicle diameters, concentrations of E2 and the ratio of E2 to progesterone (E/P) in the follicular fluid. The categories were as follows: small follicles (mean diameter: 7.4 mm, SF), estrogen-inactive dominant follicles (mean diameter: 13.5 mm, E/P < 1: EID with stages I–III CL), estrogen active dominant follicles (mean diameter: 13.5 mm, E/P ≥ 1: EAD with stages I–III CL) and preovulatory follicles (mean diameter: 16.8 mm, E/P ≥ 1: POF with stage IV CL). Each group consists of five follicles obtained from five different cows. Follicular walls were carefully peeled off from the ovaries and individually placed in the RNA later and frozen at −30 ◦ C.
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The follicular walls were cut 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 much GCs as possible. Surrounding stroma and theca externa were also removed from the follicular walls, and the cleaned follicular walls were used as TCs. 2.3. Granulosa cell culture Ovaries were obtained from cows and heifers (Holstein breed) at a local slaughterhouse. Granulosa cells were collected from follicles (5–6 mm) by aspiration and filtrated through a stainless steel filter (45 m, TOKYO SCREEN CO., LTD., Tokyo, Japan) to remove oocytes. Then, the cells were centrifuged at 800 × g and washed twice in culture medium; DMEM/F12 medium containing amphotericin B (10 l/ml) and gentamicin (5 l/ml). The cells were cultured in culture medium containing 10% FCS for 24 h at 37 ◦ C in 5% CO2 and then the wells were washed with PBS to remove unattached cells. To examine the expression of APJ on granulosa cell apoptosis, the culture medium was replaced with serum-free medium supplemented with or without 100 ng/ml FSH and the cultures were continued for 48 h. To examine the effect of steroids on the expression of the APJ gene, the culture medium was replaced with serum-free medium supplemented with oestradiol-17 (1 ng/ml or 10 ng/ml; Sigma Chemical Co., St. Louis, MO, USA), progesterone (P4, 1–100 ng/ml; Sigma Chemical Co., St. Louis, MO, USA) at various concentrations, and the culture was continued for 6 h. The treatments were terminated by aspirating medium and rinsing the cells twice with phosphate-buffered saline (Sigma Chemical Co., St. Louis, MO, USA), and were then stored in TRIZOL® at −80 ◦ C until used for RNA extraction. The replication for culture experiment performed three experiments with triplicate in each. 2.4. Hoechst 33342 staining Hoechst staining was performed to confirm the apoptotic profile as a result of morphological changes in the nucleus in which Hoechst 33342 binds specifically to the A–T base region in DNA and emits fluorescence. The cultured cells were rinsed in PBS and fixed with 1% glutaraldehyde (Wako) in PBS at 4 ◦ C for 24 h. Then the cells were stained with Hoechst 33342 (50 g/l) in PBS for 3 min. The proportion of cells with nuclear fragmentation was calculated by counting the number of stained cells per 200 cells. 2.5. Theca cell culture Ovaries were obtained from cows and heifers (Holstein breed) at a local slaughterhouse. Theca cells were isolated from the ovaries using the following method (Murayama et al., 2008). Briefly, large follicles (>10 mm in diameter) were selected and follicular fluid was aspirated using a syringe with a 22-gauge needle. The follicles were opened by making a small incision on the surface. Granulosa cells were removed by gentle scraping with a medicine spoon under a stereomicroscope. We checked the complete removal of granulosa cells under a stereomicroscope. The thecal layer was placed into PBS containing 2 mg/ml collagenase (452 U/mg, type 1, Sigma), 1 mg/ml hyaluronidase (391 U/mg, type VIII, Sigma), 1 mg/ml protease (4.5 U/mg, Sigma), and 0.4% (v/v) bovine serum albumin, and the dissociation reaction was performed for 40 min at 37 ◦ C. Centrifugal separation was carried out by 350 × g. Then, Tris–HCl buffer (pH 8.0) was put into the tube for 1 min at 37 ◦ C. The dispersed cells were washed twice with PBS. Theca cells were seeded at a density of 1 × 105 cells per well (24-well culture plate) in 1 ml of Dulbecco’s modified Eagle’s /F12 medium (DMEM/F12; Sigma Chemical Co.) containing amphotericin B (10 l/ml), gentamicin (5 l/ml), and 5% fetal calf serum (FCS; Biowest, Rue de la Caille, Muaille, France) as a pre-incubation for 24 h. After pre-incubation, the culture medium was replaced with serum-free medium supplemented with or without 100 ng/ml LH, and the cultures were continued for 24 h and 48 h. The treatments were terminated by aspirating medium and by rinsing the cells
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twice with phosphate-buffered saline, and were then stored in TRIZOL® at −80 ◦ C until used for RNA extraction. 2.6. RNA extraction and reverse transcriptase (RT) Follicle tissue samples were homogenized in denaturing solution containing 4 M guanidium thiocyanate (Wako Pure Chemical Industries Ltd., Osaka, Japan), 25 mM sodium citrate (Wako), 0.5% sarkosyl (Sigma), and 0.1 M beta-mercapto ethanol (Kanto Chemical Co. Inc., Tokyo, Japan). Total RNA was extracted with phenol–chloroform, purified further, 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 Inc., TX, USA). In the cultured samples total RNA was extracted with TRIZOL® regent (Invitrogen Japan K.K.) following the method provide by the manufacturer and frozen at −20◦ C in RNA storage solution. Single-strand cDNA was reverse transcribed from total RNA (500 ng) using a 1st Strand cDNA Synthesis Kit for RT-PCR (Roche Diagnostics Co., Indianapolis, IN, USA) and random primer. 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. 2.7. Quantitative PCR Abundances of mRNA for apelin and the APJ receptor were quantified by real-time PCR with iQ5Cycler (Bio-Rad Laboratories, CA, USA) using a commercial kit (QuantiTectTM SYBR® Green PCR: QIAGEN GmbH, Hilden, Germany). The primers used for an amplification of the apelin, APJ receptor, and -actin mRNA were as follows: apelin (106 bp) forward: 5 -aag gca cca tcc gat acc tg-3 , reverse: 5 -atg gga ccc ttg tgg gag a-3 ; APJ receptor (100 bp) forward: 5 -tct ggg cca cct aca cct at-3 , reverse: 5 -acg ctg gcg tac atg ttg-3 ; -actin (256 bp) forward: 5 -cca agg cca acc gtg aga aga t-3 , reverse: 5 -cca cgt tcc gtg agg atc ttc a-3 . The amplification program consisted of 15 min for activation at 95 ◦ C followed by 40 cycles of PCR (94 ◦ C for 15 s, specific annealing temperature for each factor for 30 s and 72 ◦ C for 20 s). 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. The PCR products were subjected to electrophoresis, and the target band was cut out and purified using a DNA purification kit (SUPRECTM -01, TaKaRa Bio. Inc., Otsu, Japan) for DNA standard. Five to seven serially diluted DNA standards were included in every PCR run. Standard curve was obtained with several dilutions of target genes (apelin and APJ) from crossing points (cycle numbers) plotted against the logarithmic concentration of the serial dilutions. The values were normalized using -actin as the internal standard. -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 and Dias, 1993). 2.8. Data analysis All data are presented as mean ± S.E.M. The level of several factors in the follicles at different developmental stages and the treated bovine granulosa cells or theca cells was tested for significant differences using ANOVA, followed by the Tukey–Kramer test as a multiple comparison test. The differences of expression of the APJ receptor in cultured granulosa cells with or without FSH were analyzed by the Student’s t-test. Differences were considered significant at p < 0.05 or less. 3. Results 3.1. Expression of apelin and APJ mRNA in granulosa cells The expression of apelin mRNA was not found in the granulosa cells of follicles at different developmental stages (Fig. 1A). On the other hand, expression of the APJ gene was significantly
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Fig. 1. (A) Representative electrophoresis image and (B) quantitative expression of APJ receptor mRNA in granulosa cells of small (SF, n = 5), estradiol inactive dominant (EID, n = 5), estradiol active dominant (EAD, n = 5), and preovulatory follicles (POF, n = 5). Each group consists of five follicles obtained from five different cows. Different superscripts denote significantly different values (p < 0.05). M shows a molecular marker (100 bp ladder).
increased in granulosa cells of estrogen-inactive dominant follicles compared with others (Fig. 1A and B). 3.2. The effect of progesterone and estradiol on the expression of APJ in granulosa cells We found a high expression of APJ mRNA in the estrogen-inactive dominant follicles compared with other follicles. The ratio of E2/P4 of EID follicles is lower level than one. Therefore, we examined the effect of progesterone alone and the combination of progesterone and estradiol on the expression of APJ mRNA in the granulosa cells. The level of APJ mRNA in cells treated with 1 ng/ml progesterone plus 10 ng/ml estradiol, or 10 ng/ml progesterone plus 1 ng/ml estradiol was increased compared with the levels in those treated with 1 ng/ml or 10 ng/ml progesterone alone (Fig. 2A and B). 3.3. Granulosa cell apoptosis and APJ expression Since APJ receptor expression is high in estrogen-inactive dominant follicles which undergo atresia (granulosa cell apoptosis), we examined the relationship between granulosa cell apoptosis and APJ expression in cultured granulosa cells. The incidence of granulosa cell apoptosis was determined by Hoechst 33342 nuclear staining in granulosa cells. FSH suppressed the granulosa cell apoptosis when the cells are cultivated in serum-free medium (Fig. 3A), and inhibited the expression of APJ receptor in the granulosa cells at 48 h of vitro culture (Fig. 3B). 3.4. Expression of apelin and APJ mRNA in theca tissues The expression of apelin mRNA significantly increased in theca tissues of estrogen-inactive dominant follicles compared with others (Fig. 4A and B). Except estrogen-inactive dominant follicles, APJ expression increased with follicle growth (Fig. 4C).
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Fig. 2. Effects of progesterone (A) and the combination of progesterone and estradiol (B) on the expression of APJ receptor mRNA in cultured granulosa cells. The data are shown as mean ± S.E.M. of three experiments with triplicate determination in each. Different superscripts denote significantly different values.
3.5. The effect of LH on APJ and apelin expression in theca cells Since a large number of factors derived in theca cells are influenced by LH, we examined the effect of LH on the expression of apelin and APJ receptor mRNAs in theca tissues. LH stimulated the expression of apelin and APJ receptor mRNAs at 24 h and 48 h of culture period, respectively (Fig. 5A and B). 4. Discussion In the mammalian ovary, the follicle acquires a vascular wreath in the theca layer which, when fully established, consists of two concentric networks of vessels in the theca interna and externa, respectively (Reynolds et al., 1992; Findlay, 1986; Gordon et al., 1995; Augustin et al., 1995; Goede et al., 1998). The establishment of the vascular wreath, particularly the expansion of the inner capillary plexus of the theca interna, coincides with a period of rapid growth and differentiation of the follicle. The differences in microvasculature in the thecal layer of follicles are an important underlying cause of follicular heterogeneity such as dominance or atresia. Such vascular formation in the theca layer during follicular development is regulated by angiogenic factors (Shimizu et al., 2003). Recently, it has been reported that both apelin and the APJ receptor are expressed in the vascular system (Devic et al., 1999; Kleinz and Davenport, 2004). Our present study indicated that the APJ receptor is expressed in granulosa cells, and that both apelin and the APJ receptor are expressed in the theca cell layer of follicles in bovine ovaries. In addition, we found that the expression of APJ receptor mRNA increased in theca tissue of POF, in which a large amount of blood vessels exist in the theca tissue, compared with those of SF. However, apelin expression in granulosa cells decreased in POF and EAD follicles
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Fig. 3. FSH suppresses granulosa cell apoptosis. (A) The granulosa cells were cultured with 100 ng/ml of FSH for 24 h and 48 h. (B) FSH suppressed the expression of APJ receptor in culture granulosa cells for 48 h. Each data point represents the mean ± S.E.M. of three experiments with triplicate determinations. An asterisk denotes a significant difference at p < 0.05.
than in EID follicles. Therefore, our findings suggested that the apelin/APJ system may not strongly be associated with thecal vasculature during follicular development in the bovine ovary. In this study, APJ receptor mRNA, but not apelin mRNA, was expressed in granulosa cell of follicles at different developmental stages. Although the EAD and EID follicles that were used in the present study had the same diameter, the APJ receptor mRNA expression in the granulosa cells of EID follicles was higher than that in the EAD follicles. The EID follicles in the present study represent the period of decreasing estradiol and increasing progesterone in the environment. Such a follicle undergoes atresia, which is induced by granulosa cell apoptosis (Tilly et al., 1991; Hsueh et al., 1994; Liu et al., 2003). We examined the effect of progesterone and estradiol on the expression of APJ receptor mRNA in granulosa cells. The expression of APJ receptor mRNA is induced by progesterone, which is present in high levels in the follicular fluid of atretic follicles, and was not suppressed when estradiol was added to the culture medium. Moreover, we examined the involvement of AJP receptor expression and granulosa cell apoptosis using an in vitro culture system. The expression of APJ mRNA significantly increased when granulosa cell apoptosis was induced. These data suggest that the APJ receptor may be associated with the follicular atresia induced by granulosa cell apoptosis during bovine follicular development. Therefore, the results from this study indicate that the increase in expression of APJ receptor mRNA in granulosa cells may be one of the characteristics of follicular atresia. We found that mRNA of both apelin and the APJ receptor are expressed in the theca cell layer of bovine follicles. Interestingly, mRNA expression of apelin is increased in the theca cell layer of EID follicles compared with those of others. In addition, we observed that the expression of APJ receptor
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Fig. 4. Expression of (A) apelin and (B) APJ receptor mRNA in theca tissues of small (SF, n = 5), estradiol inactive dominant (EID, n = 5), estradiol active dominant (EAD, n = 5), and preovulatory follicles (POF, n = 5). Each group consists of five follicles obtained from five different cows. Different superscripts denote significantly different values (p < 0.05).
mRNA was high in the granulosa cells of EID follicle. Therefore, theca cell-derived apelin may be involved in atresia by inducing granulosa cell apoptosis. In a future study we plan to examine the effect of apelin on granulosa cell apoptosis. In this study, although the expression of APJ receptor mRNA in theca tissues increased as follicle grew, apelin expression in those tissues of the follicles except EID follicles was the constant level. Thus, these results suggested that the increase in the expression of APJ receptor mRNA in theca tissues may be associated with bovine follicular development, and that the apelin–APJ system may influence the function of theca cell by autocrine manner. We found that FSH suppressed the expression of APJ receptor mRNA in cultured granulosa cells. In the present study the expression of APJ receptor mRNA was low in the granulosa cells of SF, EAD, and POF compared with EID follicles. Therefore, FSH may contribute to the maintenance of healthy follicles by inhibiting APJ receptor during follicular development. To examine the effect of gonadotropin on the expression of apelin and APJ receptor in theca cells, we evaluated the effect of LH on the expression of apelin and APJ receptor mRNAs by using cultured theca cells. LH induced the expression of apelin and the APJ receptor in theca cells at 24 h and 48 h of culture, respectively. Although the expression pathway for apelin and APJ by LH is still unknown, the different expression timing on those factors may depend on the transcription pathway. LH is essential to stimulate antral follicles to grow beyond 9 mm in diameter when a shift from FSH- to LH-dependency has taken place (Gong et al., 1996). The mean diameter of EAD and POF follicles in the present study was 13.5 mm and 16.8 mm, respectively, and these follicles have a high sensitivity to LH. Therefore, our results suggested that the apelin/APJ system in theca tissue may play an important role for in follicle growth to the ovulatory phase. In conclusion, the present study provides the first evidence that apelin in granulosa cells, and apelin and APJ in theca tissue are expressed in bovine follicles. Our findings demonstrate that the APJ receptor in granulosa cells may be associated with follicular atresia, and that aplein and APJ expression in the
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Fig. 5. Effect of LH on the expression of (A) apelin and (B) APJ receptor mRNAs in cultured theca cells. The data are shown as mean ± S.E.M. of three experiments with triplicate determination in each. An asterisk denotes a significant difference at p < 0.05.
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