- Email: [email protected]
Immunohistochemical detection of insulin-like growth factor-I, transforming growth factor-β2, basic fibroblast growth factor and epidermal growth factor-receptor expression in developing rat ovary
Cytokine 43 (2008) 209–214
Contents lists available at ScienceDirect
Cytokine journal homepage: www.elsevier.com/locate/issn/10434666
Immunohistochemical detection of insulin-like growth factor-I, transforming growth factor-b2, basic fibroblast growth factor and epidermal growth factor-receptor expression in developing rat ovary K. Ergin a,*, E. Gürsoy a, Yücel Basßımog˘lu Koca c, H. Basßalog˘lu b, K. Seyrek d a
Department of Histology and Embryology, Adnan Menderes University School of Medicine, TR-09100 Aydın, Turkey Department of Anatomy, Adnan Menderes University School of Medicine, TR-09100 Aydın, Turkey c Department of Biology, Adnan Menderes University Science and Arts, TR-09100 Aydın, Turkey d Department of Biochemistry, Adnan Menderes University School of Veterinary Medicine, TR-09100 Aydın, Turkey b
a r t i c l e
i n f o
Article history: Received 6 June 2007 Received in revised form 14 April 2008 Accepted 14 May 2008 Keywords: Growth factor Follicle Ovary Immunohistochemistry
a b s t r a c t The aim of this study was to determine the immunohistochemical expression and localization of insulinlike growth factor-I (IGF-I), transforming growth factor-b2 (TGF-b2), basic fibroblast growth factor (bFGF) and epidermal growth factor-receptor (EGF-R) in developing rat ovaries. Eighteen female Wistar rats were enrolled in this study; newborn (n = 6), one-month-old (n = 6) and adult (n = 6) rats. Formalin-fixed and parafin-embedded ovarian tissues were stained with antibodies against IGF-I, TGF-b2, bFGF and EGF-R, immunohistochemically. The ovarian cells were evaluated by semi-quantitative scoring system under light microscope. The staining of IGF-I, TGF-b2, bFGF and EGF-R were most intense in the oocytes and were heavily at onemonth-old rats. A moderate immunostaining in theca cells and corpus luteii reacted with IGF-I in adult rats. Furthermore the staining intensity for IGF-I was moderate in granulosa cells of newborn rat ovaries. We detected also a moderate staining for TGF-b2 in corpus luteii of adult rats. In addition, we found a bFGF immunostaining mainly in oocytes of follicles of young and adult rats. Immunostaining for EGF-R was moderate in granulosa cells of one-month-old rats. In conclusion, this study suggests that growth factors play a pivotal role in ovarian function, especially in follicular development. The role of growth factor in controlling degeneration or growth (or both) of ovary follicles remain as explained. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction More than 99% of follicles in ovary undergo atresia, during the reproductive life of the females [1]. Atresia occurs by apoptosis of the follicular cells and of the oocytes [2]. Successful follicle development depends on the presence of survival factors that protect cells from apoptosis and also promote follicle growth in ovary [2]. Evidence suggest that growth factors influence ovarian physiology in different stages of follicular growth via endocrine, paracrine and autocrine mechanisms [3,4]. Growth factors seem to modulate cell survival and cell death balance between the granulosa cells [5] and play an important role in the regulation of ovarian follicular development [2,6,7]. It was shown in several mammalian species including rats that insulin-like growth factor-I (IGF-I) stimulates both proliferation and differentiation of granulosa cells [2,6,7] and promotes granu-
* Corresponding author. Fax: +90 256 2123169. E-mail address: [email protected] (K. Ergin). 1043-4666/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.cyto.2008.05.013
losa cell survival by increasing resistance to apoptosis [7]. IGF-I, basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) seem to have an important anti-apoptotic effect in ovarian tissues [5,8,9]. It was also reported that IGF-I, bFGF and EGF support growth of small antral follicles by enhancing granulosa cell proliferation [10]. IGF-I, EGF and transforming growth factor-beta (TGF-b) have also been shown to be involved in ovarian follicular dynamics [4,11]. It is also believed that bFGF have an important role in early folliculogenesis and can regulate primordial development [12,13]. In addition, it has been shown that TGF-b was crucial for embryogenesis and for regulation of cellular activities such as proliferation and apoptosis, which are important for tissue growth and morphogenesis [14,15]. Multiple studies have been performed in various mammalian species to demonstrate the roles for IGF-I, TGF-b, bFGF and EGF-R proteins in various aspects of follicular growth and ovarian development. However, to our knowledge, there is no study that compares the expression and localization of IGF-I, TGF-b2, bFGF and EGF-R proteins in ovaries of newborn, one-month-old and adult rats. Thus, the aim of this study was to determine the expression
210
K. Ergin et al. / Cytokine 43 (2008) 209–214
and localization of IGF-I, TGF-b2, bFGF and EGF-R in the stage of developing rat ovary.
Table 2 Expression and distribution of IGF-I, TGF-b2, bFGF and EGF-R in the stroma and corpus luteum of developing rat ovary IGF-I
TGF-b2
bFGF
EGF-R
Table 1 Expression and distribution of insulin-like growth factor-I (IGF-I), transforming growth factor-beta 2 (TGF-b2), fibroblast growth factor-2 (FGF-2) and epidermal growth factor receptor (EGF-R) in the follicles of developing rat ovary
Stroma Newborn One-month- old Adult
+++ +++
++ ++
+
+
Corpus luteum Adult
++
++
+
Primordial follicles IGF-I Newborn Granulosa cells Oocyte Theca cells One-month-old Granulosa cells Oocyte Theca cells Adult Granulosa cells Oocyte Theca cells TGF-b2 Newborn Granulosa cells Oocyte Theca cells One-month-old Granulosa cells Oocyte Theca cells Adult Granulosa cells Oocyte Theca cells bFGF (FGF-2) Newborn Granulosa cells Oocyte Theca cells One-month-old Granulosa cells Oocyte Theca cells Adult Granulosa cells Oocyte Theca cells EGF-R Newborn Granulosa cells Oocyte Theca cells One-month-old Granulosa cells Oocyte Theca cells Adult Granulosa cells Oocyte Theca cells
Primary follicles
Secondary follicles
Tertiary follicles
The intensity of staining is grouped into categories: , no staining; +, weak staining; ++, medium staining; +++, strong staining. ++
++
Not available
Not available
+
Not available
+++
+++
+++
+
++ ++
+ ++
Not available
Not available
+
+
+++ +
++
++
Not available
Not available
Not available
+++
+++
++
++
Not available
Not available
+++
+++
+
++
++
Not available
+
++
++
+
+
+
+
+
+
2. Material and methods 2.1. Animals and treatment Newborn (n = 6), one monthly (n = 6) and adult (n = 6) female Wistar rats were used in this study. They were kept at a constant temperature (21 ± 1 °C) and controlled light conditions (light 07.00–19.00). Food (standard pellet diet) and tap water were supplied ad libitum. Rats were anesthetized with ether and were killed by cervical dislocation for collection of ovary. All studies with animals described here in, were reviewed and approved by the University of Adnan Menderes Animal Ethics committee. 2.2. Immunolocalization Not available
The intensity of staining is grouped into categories: , no staining; +, weak staining; ++, moderate staining; +++, strong staining.
Ovary samples were fixed in 10% neutral buffered formalin, routinely processed (dehydration steps in ethanol and clearing in xylene) and embedded in paraffin blocks. Tissues in paraffin blocks were randomly cut in 5 lm sections by a microtome (Leica RM 2135) and placed on poly-L-lysine coated glass slides. Immunohistochemical staining was performed by avidin–biotin immunoperoxidase system. After 2 h incubation at 40 °C, sections were deparaffinized in xylene, hydrated through graded ethanol and endogenous peroxidase blocked with 3% H2O2 in 70% methanol. The sections were washed as in step 3 for 10 min in phosphatebuffered-saline (PBS, pH 7,3), and nonspecific protein-binding sites were blocked with 3% normal goat serum (Camon, Wiesbaden, Germany) to reduce background staining. The sections were processed for immunlocalization of IGF-I, TGF-b2, bFGF (FGF-2), and EGF-R using a commercially available polyclonal antibody against IGF-I (1:50 dilution; Labvision Corporation, Suffolk, UK), a polyclonal antibody against TGF-b2 (1:50 dilution; Labvision Corporation, Suffolk, UK), a polyclonal antibody against bFGF (1:200 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) and a polyclonal antibody against EGF-R (1:200 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) as the primary antibodies. The optimum working dilutions were determined by serial titration. After washing with PBS, the specimens were incubated with biotinylated goat anti-polyvalent secondary antibody (Labvision Corporation, Suffolk, UK) for IGF-1, TGF-b2 EGF-R and bFGF, for 30 min at room temperature. Streptomycin avidin-peroxidase conjugate (Labvision Corporation, Suffolk, UK) was added and incubated for 30 min at room temperature. The sections were visualized using 0.05% (w/v) 3,30 -diaminobenzidine and 0.010% (v/v) hydrogen peroxide in PBS (10 mM, pH 7.4). These sections were counter-stained with hematoxylin and mounted with entellan. Control procedure were undertaken to insure the specificity of immunoreaction. Negative controls were carried out by replacing the primary antibodies with PBS. Screen shots were taken with Olympus Digital camera (DP 20) attached at Olympus BX51 microscope. All the slides were examined by the same observer who was blind to the tissue sections
K. Ergin et al. / Cytokine 43 (2008) 209–214
between the groups. The intensity of immunohistochemical staining was graded semi-quantitatively as follows: (–) no immunostaining; (+) weak staining; (++) moderate staining; (+++) strong staining. 3. Results The localization of IGF-I, TGF-b2, bFGF and EGF-R proteins in cellular compartments in follicles is shown in Table 1 and 2. Granulosa cells of the primordial and primary follicles of newborn rat ovaries showed a moderate staining for IGF-I (Fig. 1A), while a strong staining for EGF-R in oocytes (Fig. 4A) was observed. However no staining for TGF-b2 and bFGF in oocytes and granulosa cells (Figs. 2 and 3A) was observed. When compared to newborn rats, a weak staining in granulosa cells of primary follicles and no staining in granulosa cells of secondary follicles for IGF-1 was observed in one-month-old rat ovaries. Primordial and primary follicles showed a strong staining for IGF-I in oocytes (data not shown) as well as secondary follicles
211
(Fig. 1B). In addition a strong TGF-b2 and bFGF staining was seen in oocytes of the secondary follicles (Figs. 2 and 3B) and a moderate EGF-R staining in oocytes and granulosa cells of primary follicles (data not shown) and secondary follicles (Fig. 4B) was observed in one-month-old rats. In comparison to one-month-old rats, oocytes of adult rat ovaries showed a decreased staining for IGF-I (Fig. 1C), while a moderate IGF-I staining in theca cells of the secondary and tertiary follicles (Fig. 1C) was observed. Besides a moderate TGF-b2 staining in oocytes of primary follicles (data not shown) and secondary follicles (Fig. 2C) was observed. Oocytes of the secondary follicles (Fig. 3C) and tertiary follicles (data not shown) showed a moderate staining for bFGF in the adult group. Granulosa cells and oocytes of adult rat ovaries showed a weak staining for EGF-R (Fig. 4C). The stroma in one-month-old (Fig. 5A) and adult group (data not shown) showed a strong staining for IGF-I, while a moderate staining for TGF-b2 in one-month-old (data not shown) and adult group (Fig. 5B) was observed. A weak staining for bFGF in the one-month-old group (Fig. 5C), and a weak staining for EGF-R in the adult group (Fig. 5D) was observed. The corpus luteii in the
Fig. 1. IGF-I; (A) Newborn rat, moderate staining in granulosa cells of primary and primordial follicles (arrowheads), original magnification: 400 (scale bars, 20 lm), (B) onemonth-old rat, strong staining in the oocyte of a secondary follicle (asterisk), original magnification: 400 (scale bars, 20 lm), (C) Adult rat, weak staining in the oocyte (asterisk) and a moderate staining in theca layer of a tertiary follicle (arrow), original magnification: 200 (scale bars, 40 lm).
Fig. 2. TGF-b2; (A) Newborn rat, no staining in follicles, original magnification: 400 (scale bars, 20 lm). (B) One-month-old rat, strong staining in the oocyte of a secondary follicle (asterisk), original magnification: 400 (scale bars, 20 lm), (C) Adult rat, moderate staining in the oocyte of a secondary follicle (asterisk), original magnification: 200 (scale bars, 40 lm).
Fig. 3. bFGF; (A) Newborn rat, no staining in follicles, (B) One-month-old rat, strong staining in the oocyte of a secondary follicle (asterisk), (C) Adult rat, moderate staining in the oocyte of a secondary follicle (asterisk). Original magnification: 400 (scale bars, 20 lm).
212
K. Ergin et al. / Cytokine 43 (2008) 209–214
Fig. 4. EGF-R; (A) Newborn rat, strong staining in oocytes of primary follicles (arrowheads), original magnification: 400 (scale bars, 20 lm), (B) one-month-old rat, moderate staining in the oocyte of a secondary follicle (asterisk), original magnification: 400 (scale bars, 20 lm), (C) Adult rat, weak staining in granulosa cells (arrowheads) and in the oocyte (asterisk) of a tertiary follicle. Original magnification: 200 (scale bars, 40 lm).
Fig. 5. Stroma cells; (A) Strong staining for IGF-I (asterisks) in one-month-old rat ovary, (B) Moderate staining for TGF-b2 (asterisks) in adult rat ovary, (C) Weak staining for bFGF (asterisk) in one-month-old rat ovary, (D) Weak staining for EGF-R (asterisks) in adult rat ovary. Original magnification: 100 (scale bars, 80 lm).
adult group showed a moderate staining for IGF-I (Fig. 6A) and for TGF-b2 (Fig. 6B). However, no staining for bFGF and a weak staining for EGF-R was observed (Fig. 6C and D). 4. Discussion In this study, we intended to detect the expression and localization of IGF-I, TGF-b2, bFGF and EGF-R in rat ovaries of different ages by immunohistochemistry. It is reviewed that IGF-I stimulated proliferation or differentiation of granulosa cells, depending on the stage of development of the follicle and it also stimulates proliferation of granulosa cells from small follicles [16]. In addition it was claimed that IGF family
plays an important role in follicle development, dominant follicle growth, steroidogenesis and follicular atresia [17]. Previous studies reported that IGF-I regulates human luteal steroidogenesis by an increase in progesterone [18] and oestradiol production [19]. IGFI was regarded as a co-gonadotrophin of LH in stimulating steroidogenesis in theca-intertitial cells [20]. In this study we found a moderate staining for IGF-I in granulosa cells of newborn rats, a strong staining in oocytes of young rats, a strong staining in stroma cells of young and adult rats, a moderate staining in theca cells of adult rats and also a moderate staining persisted in the corpus luteii. As it was seen, IGF-1 affected different parts of the ovary follicles in different ages. Hormonal changes with increasing age and development of the follicles might be the cause of these findings.
K. Ergin et al. / Cytokine 43 (2008) 209–214
213
Fig. 6. Corpus luteii in the adult group (A) Moderate staining for IGF-I, (B) Moderate staining for TGF-b2, (C) No staining for bFGF, (D) Weak staining for EGF-R. Original magnification: 200 (scale bars, 40 lm).
One family of peptides known to be involved in the regulation of ovarian development and function is the TGF-b superfamily [11]. Liu et al. were found that TGF-b increases the diameter of follicles from adult mice and has no effect on preantral follicles from immature mice [21]. TGF-b did not have an effect on preantral follicles from 11-day-old mice [21]. On the other hand, TGF-b stimulated the follicular growth of 56-day-old mice [21]. Interestingly, preantral follicles of 28-day-old mice showed an intermediate response, suggesting that the different responses are related to the process of physical maturity [21]. In comparison of newborn, young and adult rats, we observed TGF-b staining only in oocytes of young and adult rats in a variety of degrees. As a result of these findings, we thought that the effect of TGF-b does not appear until puberty. It is reviewed by Knight et al. that members of the TGF-b superfamily, are expressed in oocytes in a developmental-stage related manner and act as intraovarian regulatory molecules [22]. Juengel and McNatty reported that TGF-b2 was found in the oocytes of secondary follicles in rodents [23]. Similarly, we found stronger TGFb2 staining in the oocytes of secondary follicles in young and adult rats. We suggested that TGF-b actions may be age and stage dependent. In the study of Chegini and Flanders, only small luteal cells showed intense immunostaining for TGF-b2; however, the large luteal cells had a weak immunostaining at midluteal phase in human ovary [24]. Besides, ovarian stromal cells do not immunostain for TGF-b2 [24]. In this study, we found a moderate staining of TGF-
b2 in the cells of the corpus luteii and of the stroma. The difference of staining intensity might be due to different sizes of corpus luteii cells. Thus it could be the limitation of our study, not to focus on the sizes of corpus luteii cells and stages of the luteal phase. On the other hand, compared to Chegini and Flanders’ study, the difference of staining intensity in stromal cells might be because of using rat ovaries via human ovaries. In this respect, we could suggest that TGF-b2 may affect the oocyte development and corpus luteum survival. The difference between the species might affect the expression of growth factors such as bFGF. Ben-haroush et al. were detected bFGF in granulosa cells from primary follicular stages onward of adolescents/women, and in oocytes from fetuses and adults at all follicular classes [12]. Besides Nilsson et al. were found that bFGF was localized to primordial and early developing follicles and were mainly observed in the oocytes of 4-day-old rat ovaries which were isolated and cultured for 14 days. In that study, elevated levels of bFGF in the oocytes of primordial and early developing follicles was described [13]. In our study, we found strong immunostaining in oocytes of primary and secondary follicles of young rats, moderate staining in oocytes of secondary and tertiary follicles of adult rats and no staining in newborn rats. Because we used different age groups of rats such as newborn (0-day), young and adults, and studied only immunohistochemically, our findings were not in accordance with Nilsson’s study. In addition to this, our findings
214
K. Ergin et al. / Cytokine 43 (2008) 209–214
showed that bFGF affected follicles especially in young and adult rats. In some studies EGF-receptor (EGF and TGFa bind to this receptor) has been identified in granulosa cells, corpus luteum and oocytes [25,26]. Dark staining was found in the oocytes compared to follicular and stromal cells in human fetuses [25]. Bennet et al. postulated that EGF-R may act as local regulators for the development or degeneration of individual oocytes [25]. We found a strong staining for EGF-R in oocytes of newborn rats, a moderate staining in one-month-old rats and a weak staining in adult rats. From this point of view, a decreased staining of EGF-R was seen by increasing age. Therefore, we thought that the effect of EGF-R were decreasing with age and hence EGF-R might have the strongest effects in newborn term. Ayyagari and Khan-Dawood demonstrated the presence of EGFreceptors in human corpus luteum [26]. We found a weak staining in corpus luteii for EGF-R. Since EGF-R staining decreased with age in oocytes, this was an expected result. In conclusion, we have demonstrated the presence of IGF-I, TGFb2, bFGF and EGF-R proteins in developing ovaries, suggesting that growth factors play a pivotal role in ovarian function. The role of growth factors in controlling degeneration or growth (or both) of ovary follicles remain as explained. Therefore, additional studies should be undertaken to elucidate the role of these and other proteins in development and atresia of ovarian follicles. Acknowledgments The authors thank the Adnan Menderes University Research Fund for financial support (TPF-4015) for this study and Assist. Prof. Dr. Yüksel Yıldız for his language assistance. References [1] Hsueh AJ, Billig H, Tsafriri A. Ovarian follicle atresia: a hormonally controlled apoptotic process. Endocr Rev 1994;15:707–24. [2] Quirk SM, Cowan RG, Harman RM, Hu CL, Porter DA. Ovarian follicular growth and atresia: the relationship between cell proliferation and survival. J Anim Sci 2004;82(E-Suppl):E40–52. [3] Kaiser GG, Sinowatz F, Palma GA. Effects of growth hormone on female reproductive organs. Anat Histol Embryol 2001;30:265–71. [4] Ulug U, Turan E, Tosun SB, Erden HF, Bahceci M. Comparison of preovulatory follicular concentrations of epidermal growth factor, insulin-like growth factor-I, and inhibins A and B in women undergoing assisted conception treatment with gonadotropin-releasing hormone (GnRH) agonists and GnRH antagonists. Fertil Steril 2007;87:995–8. [5] Luciano AM, Modina S, Gandolfi F, Lauria A, Armstrong DT. Effect of cell-to-cell contact on in vitro deoxyribonucleic acid synthesis and apoptosis responses of bovine granulosa cells to insulin-like growth factor-I and epidermal growth factor. Biol Reprod 2000;63:1580–5. [6] Monget P, Fabre S, Mulsant P, Lecerf F, Elsen JM, Mazerbourg S, et al. Regulation of ovarian folliculogenesis by IGF and BMP system in domestic animals. Domest Anim Endocrinol 2002;23:139–54.
[7] Mazerbourg S, Bondy CA, Zhou J, Monget P. The insulin-like growth factor system: a key determinant role in the growth and selection of ovarian follicles? a comparative species study. Reprod Domest Anim 2003;38:247–58. [8] Villavicencio A, Iñiguez G, Johnson MC, Gabler F, Palomino A, Vega M. Regulation of steroid synthesis and apoptosis by insulin-like growth factor I and insulin-like growth factor binding protein 3 in human corpus luteum during the midluteal phase. Reproduction 2002;124(4):501–8. [9] Nikolettos N, Asimakopoulos B, Köster F, Schöpper B, Scultz C, Caglar GS, et al. Cytokine profile in cases with premature elevation of progesterone serum concentrations during ovarian stimulation. Physiol Res 2007;8. [Epub ahead of print]. [10] Monniaux D, Monget P, Besnard N, Huet C, Pisselet C. Growth factors and antral follicular development in domestic ruminants. Theriogenology 1996;47:3–12. [11] Drummond AE. TGF beta signalling in the development of ovarian function. Cell Tissue Res 2005;322(1):107–15. [12] Ben-Haroush A, Abir R, Ao A, Jin S, Kessler-Icekson G, Feldberg D, et al. Expression of basic fibroblast growth factor and its receptors in human ovarian follicles from adults and fetuses. Fertil Steril 2005;849(Suppl 2):1257–68. [13] Nilsson E, Parrott JA, Skinner MK. Basic fibroblast growth factor induces primordial follicle development and initiates folliculogenesis. Mol Cell Endocrinol 2001;175(1–2):123–30. [14] Chen YG, Meng AM. Negative regulation of TGF-beta signaling in development. Cell Res 2004;14(6):441–9. [15] Méndez C, Alcántara L, Escalona R, López-Casillas F, Pedernera E. Transforming growth factor beta inhibits proliferation of somatic cells without influencing germ cell number in the chicken embryonic ovary. Cell Tissue Res 2006;325(1):143–9. [16] Monget P, Fabre S, Mulsant P, Lecerf F, Elsen JM, Mazerbourg S, et al. Regulation of ovarian folliculogenesis by IGF and BMP system in domestic animals. Domest Anim Endocrinol 2002;23(1–2):139–54. [17] Giudice LC. Insulin-like growth factor family in Graafian follicle development and function. J Soc Gynecol Investig 2001;8:S26–9. [18] Devoto L, Kohen P, Castro O, Vega M, Troncoso JL, Charreau E. Multihormonal regulation of progesterone synthesis in cultured human midluteal cells. J Clin Endocrinol Metab 1995;80(5):1566–70. [19] Johnson MC, Devoto L, Retamales I, Kohen P, Troncoso JL, Aguilera G. Localization of insulin-like growth factor (IGF-I) and IGF-I receptor expression in human corpora lutea: role on estradiol secretion. Fertil Steril 1996;65(3):489–94. [20] Duleba AJ, Spaczynski RZ, Olive DL, Behrman HR. Divergent mechanisms regulate proliferation/survival and steroidogenesis of theca-interstitial cells. Mol Hum Reprod 1999;5(3):193–8. [21] Liu X, Andoh K, Abe Y, Kobayashi J, Yamada K, Mizunuma H, et al. A comparative study on transforming growth factor-beta and activin A for preantral follicles from adult, immature, and diethylstilbestrol-primed immature mice. Endocrinology 1999;140(6):2480–5. [22] Knight PG, Glister C. Local roles of TGF-beta superfamily members in the control of ovarian follicle development. Anim Reprod Sci 2003;78(3–4): 165–183. [23] Juengel JL, McNatty KP. The role of proteins of the transforming growth factorbeta superfamily in the intraovarian regulation of follicular development. Hum Reprod Update 2005;11(2):143–60. [Epub 2005 Feb 10]. [24] Chegini N, Flanders KC. Presence of transforming growth factor-beta and their selective cellular localization in human ovarian tissue of various reproductive stages. Endocrinology 1992;130(3):1707–15. [25] Bennett RA, Osathanondh R, Yeh J. Immunohistochemical localization of transforming growth factor-alpha, epidermal growth factor (EGF), and EGF receptor in the human fetal ovary. J Clin Endocrinol Metab 1996;81(8):3073–6. [26] Ayyagari RR, Khan-Dawood FS. Human corpus luteum: presence of epidermal growth factor receptors and binding characteristics. Am J Obstet Gynecol 1987;156(4):942–6.
Contents lists available at ScienceDirect
Cytokine journal homepage: www.elsevier.com/locate/issn/10434666
Immunohistochemical detection of insulin-like growth factor-I, transforming growth factor-b2, basic fibroblast growth factor and epidermal growth factor-receptor expression in developing rat ovary K. Ergin a,*, E. Gürsoy a, Yücel Basßımog˘lu Koca c, H. Basßalog˘lu b, K. Seyrek d a
Department of Histology and Embryology, Adnan Menderes University School of Medicine, TR-09100 Aydın, Turkey Department of Anatomy, Adnan Menderes University School of Medicine, TR-09100 Aydın, Turkey c Department of Biology, Adnan Menderes University Science and Arts, TR-09100 Aydın, Turkey d Department of Biochemistry, Adnan Menderes University School of Veterinary Medicine, TR-09100 Aydın, Turkey b
a r t i c l e
i n f o
Article history: Received 6 June 2007 Received in revised form 14 April 2008 Accepted 14 May 2008 Keywords: Growth factor Follicle Ovary Immunohistochemistry
a b s t r a c t The aim of this study was to determine the immunohistochemical expression and localization of insulinlike growth factor-I (IGF-I), transforming growth factor-b2 (TGF-b2), basic fibroblast growth factor (bFGF) and epidermal growth factor-receptor (EGF-R) in developing rat ovaries. Eighteen female Wistar rats were enrolled in this study; newborn (n = 6), one-month-old (n = 6) and adult (n = 6) rats. Formalin-fixed and parafin-embedded ovarian tissues were stained with antibodies against IGF-I, TGF-b2, bFGF and EGF-R, immunohistochemically. The ovarian cells were evaluated by semi-quantitative scoring system under light microscope. The staining of IGF-I, TGF-b2, bFGF and EGF-R were most intense in the oocytes and were heavily at onemonth-old rats. A moderate immunostaining in theca cells and corpus luteii reacted with IGF-I in adult rats. Furthermore the staining intensity for IGF-I was moderate in granulosa cells of newborn rat ovaries. We detected also a moderate staining for TGF-b2 in corpus luteii of adult rats. In addition, we found a bFGF immunostaining mainly in oocytes of follicles of young and adult rats. Immunostaining for EGF-R was moderate in granulosa cells of one-month-old rats. In conclusion, this study suggests that growth factors play a pivotal role in ovarian function, especially in follicular development. The role of growth factor in controlling degeneration or growth (or both) of ovary follicles remain as explained. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction More than 99% of follicles in ovary undergo atresia, during the reproductive life of the females [1]. Atresia occurs by apoptosis of the follicular cells and of the oocytes [2]. Successful follicle development depends on the presence of survival factors that protect cells from apoptosis and also promote follicle growth in ovary [2]. Evidence suggest that growth factors influence ovarian physiology in different stages of follicular growth via endocrine, paracrine and autocrine mechanisms [3,4]. Growth factors seem to modulate cell survival and cell death balance between the granulosa cells [5] and play an important role in the regulation of ovarian follicular development [2,6,7]. It was shown in several mammalian species including rats that insulin-like growth factor-I (IGF-I) stimulates both proliferation and differentiation of granulosa cells [2,6,7] and promotes granu-
* Corresponding author. Fax: +90 256 2123169. E-mail address: [email protected] (K. Ergin). 1043-4666/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.cyto.2008.05.013
losa cell survival by increasing resistance to apoptosis [7]. IGF-I, basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) seem to have an important anti-apoptotic effect in ovarian tissues [5,8,9]. It was also reported that IGF-I, bFGF and EGF support growth of small antral follicles by enhancing granulosa cell proliferation [10]. IGF-I, EGF and transforming growth factor-beta (TGF-b) have also been shown to be involved in ovarian follicular dynamics [4,11]. It is also believed that bFGF have an important role in early folliculogenesis and can regulate primordial development [12,13]. In addition, it has been shown that TGF-b was crucial for embryogenesis and for regulation of cellular activities such as proliferation and apoptosis, which are important for tissue growth and morphogenesis [14,15]. Multiple studies have been performed in various mammalian species to demonstrate the roles for IGF-I, TGF-b, bFGF and EGF-R proteins in various aspects of follicular growth and ovarian development. However, to our knowledge, there is no study that compares the expression and localization of IGF-I, TGF-b2, bFGF and EGF-R proteins in ovaries of newborn, one-month-old and adult rats. Thus, the aim of this study was to determine the expression
210
K. Ergin et al. / Cytokine 43 (2008) 209–214
and localization of IGF-I, TGF-b2, bFGF and EGF-R in the stage of developing rat ovary.
Table 2 Expression and distribution of IGF-I, TGF-b2, bFGF and EGF-R in the stroma and corpus luteum of developing rat ovary IGF-I
TGF-b2
bFGF
EGF-R
Table 1 Expression and distribution of insulin-like growth factor-I (IGF-I), transforming growth factor-beta 2 (TGF-b2), fibroblast growth factor-2 (FGF-2) and epidermal growth factor receptor (EGF-R) in the follicles of developing rat ovary
Stroma Newborn One-month- old Adult
+++ +++
++ ++
+
+
Corpus luteum Adult
++
++
+
Primordial follicles IGF-I Newborn Granulosa cells Oocyte Theca cells One-month-old Granulosa cells Oocyte Theca cells Adult Granulosa cells Oocyte Theca cells TGF-b2 Newborn Granulosa cells Oocyte Theca cells One-month-old Granulosa cells Oocyte Theca cells Adult Granulosa cells Oocyte Theca cells bFGF (FGF-2) Newborn Granulosa cells Oocyte Theca cells One-month-old Granulosa cells Oocyte Theca cells Adult Granulosa cells Oocyte Theca cells EGF-R Newborn Granulosa cells Oocyte Theca cells One-month-old Granulosa cells Oocyte Theca cells Adult Granulosa cells Oocyte Theca cells
Primary follicles
Secondary follicles
Tertiary follicles
The intensity of staining is grouped into categories: , no staining; +, weak staining; ++, medium staining; +++, strong staining. ++
++
Not available
Not available
+
Not available
+++
+++
+++
+
++ ++
+ ++
Not available
Not available
+
+
+++ +
++
++
Not available
Not available
Not available
+++
+++
++
++
Not available
Not available
+++
+++
+
++
++
Not available
+
++
++
+
+
+
+
+
+
2. Material and methods 2.1. Animals and treatment Newborn (n = 6), one monthly (n = 6) and adult (n = 6) female Wistar rats were used in this study. They were kept at a constant temperature (21 ± 1 °C) and controlled light conditions (light 07.00–19.00). Food (standard pellet diet) and tap water were supplied ad libitum. Rats were anesthetized with ether and were killed by cervical dislocation for collection of ovary. All studies with animals described here in, were reviewed and approved by the University of Adnan Menderes Animal Ethics committee. 2.2. Immunolocalization Not available
The intensity of staining is grouped into categories: , no staining; +, weak staining; ++, moderate staining; +++, strong staining.
Ovary samples were fixed in 10% neutral buffered formalin, routinely processed (dehydration steps in ethanol and clearing in xylene) and embedded in paraffin blocks. Tissues in paraffin blocks were randomly cut in 5 lm sections by a microtome (Leica RM 2135) and placed on poly-L-lysine coated glass slides. Immunohistochemical staining was performed by avidin–biotin immunoperoxidase system. After 2 h incubation at 40 °C, sections were deparaffinized in xylene, hydrated through graded ethanol and endogenous peroxidase blocked with 3% H2O2 in 70% methanol. The sections were washed as in step 3 for 10 min in phosphatebuffered-saline (PBS, pH 7,3), and nonspecific protein-binding sites were blocked with 3% normal goat serum (Camon, Wiesbaden, Germany) to reduce background staining. The sections were processed for immunlocalization of IGF-I, TGF-b2, bFGF (FGF-2), and EGF-R using a commercially available polyclonal antibody against IGF-I (1:50 dilution; Labvision Corporation, Suffolk, UK), a polyclonal antibody against TGF-b2 (1:50 dilution; Labvision Corporation, Suffolk, UK), a polyclonal antibody against bFGF (1:200 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) and a polyclonal antibody against EGF-R (1:200 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) as the primary antibodies. The optimum working dilutions were determined by serial titration. After washing with PBS, the specimens were incubated with biotinylated goat anti-polyvalent secondary antibody (Labvision Corporation, Suffolk, UK) for IGF-1, TGF-b2 EGF-R and bFGF, for 30 min at room temperature. Streptomycin avidin-peroxidase conjugate (Labvision Corporation, Suffolk, UK) was added and incubated for 30 min at room temperature. The sections were visualized using 0.05% (w/v) 3,30 -diaminobenzidine and 0.010% (v/v) hydrogen peroxide in PBS (10 mM, pH 7.4). These sections were counter-stained with hematoxylin and mounted with entellan. Control procedure were undertaken to insure the specificity of immunoreaction. Negative controls were carried out by replacing the primary antibodies with PBS. Screen shots were taken with Olympus Digital camera (DP 20) attached at Olympus BX51 microscope. All the slides were examined by the same observer who was blind to the tissue sections
K. Ergin et al. / Cytokine 43 (2008) 209–214
between the groups. The intensity of immunohistochemical staining was graded semi-quantitatively as follows: (–) no immunostaining; (+) weak staining; (++) moderate staining; (+++) strong staining. 3. Results The localization of IGF-I, TGF-b2, bFGF and EGF-R proteins in cellular compartments in follicles is shown in Table 1 and 2. Granulosa cells of the primordial and primary follicles of newborn rat ovaries showed a moderate staining for IGF-I (Fig. 1A), while a strong staining for EGF-R in oocytes (Fig. 4A) was observed. However no staining for TGF-b2 and bFGF in oocytes and granulosa cells (Figs. 2 and 3A) was observed. When compared to newborn rats, a weak staining in granulosa cells of primary follicles and no staining in granulosa cells of secondary follicles for IGF-1 was observed in one-month-old rat ovaries. Primordial and primary follicles showed a strong staining for IGF-I in oocytes (data not shown) as well as secondary follicles
211
(Fig. 1B). In addition a strong TGF-b2 and bFGF staining was seen in oocytes of the secondary follicles (Figs. 2 and 3B) and a moderate EGF-R staining in oocytes and granulosa cells of primary follicles (data not shown) and secondary follicles (Fig. 4B) was observed in one-month-old rats. In comparison to one-month-old rats, oocytes of adult rat ovaries showed a decreased staining for IGF-I (Fig. 1C), while a moderate IGF-I staining in theca cells of the secondary and tertiary follicles (Fig. 1C) was observed. Besides a moderate TGF-b2 staining in oocytes of primary follicles (data not shown) and secondary follicles (Fig. 2C) was observed. Oocytes of the secondary follicles (Fig. 3C) and tertiary follicles (data not shown) showed a moderate staining for bFGF in the adult group. Granulosa cells and oocytes of adult rat ovaries showed a weak staining for EGF-R (Fig. 4C). The stroma in one-month-old (Fig. 5A) and adult group (data not shown) showed a strong staining for IGF-I, while a moderate staining for TGF-b2 in one-month-old (data not shown) and adult group (Fig. 5B) was observed. A weak staining for bFGF in the one-month-old group (Fig. 5C), and a weak staining for EGF-R in the adult group (Fig. 5D) was observed. The corpus luteii in the
Fig. 1. IGF-I; (A) Newborn rat, moderate staining in granulosa cells of primary and primordial follicles (arrowheads), original magnification: 400 (scale bars, 20 lm), (B) onemonth-old rat, strong staining in the oocyte of a secondary follicle (asterisk), original magnification: 400 (scale bars, 20 lm), (C) Adult rat, weak staining in the oocyte (asterisk) and a moderate staining in theca layer of a tertiary follicle (arrow), original magnification: 200 (scale bars, 40 lm).
Fig. 2. TGF-b2; (A) Newborn rat, no staining in follicles, original magnification: 400 (scale bars, 20 lm). (B) One-month-old rat, strong staining in the oocyte of a secondary follicle (asterisk), original magnification: 400 (scale bars, 20 lm), (C) Adult rat, moderate staining in the oocyte of a secondary follicle (asterisk), original magnification: 200 (scale bars, 40 lm).
Fig. 3. bFGF; (A) Newborn rat, no staining in follicles, (B) One-month-old rat, strong staining in the oocyte of a secondary follicle (asterisk), (C) Adult rat, moderate staining in the oocyte of a secondary follicle (asterisk). Original magnification: 400 (scale bars, 20 lm).
212
K. Ergin et al. / Cytokine 43 (2008) 209–214
Fig. 4. EGF-R; (A) Newborn rat, strong staining in oocytes of primary follicles (arrowheads), original magnification: 400 (scale bars, 20 lm), (B) one-month-old rat, moderate staining in the oocyte of a secondary follicle (asterisk), original magnification: 400 (scale bars, 20 lm), (C) Adult rat, weak staining in granulosa cells (arrowheads) and in the oocyte (asterisk) of a tertiary follicle. Original magnification: 200 (scale bars, 40 lm).
Fig. 5. Stroma cells; (A) Strong staining for IGF-I (asterisks) in one-month-old rat ovary, (B) Moderate staining for TGF-b2 (asterisks) in adult rat ovary, (C) Weak staining for bFGF (asterisk) in one-month-old rat ovary, (D) Weak staining for EGF-R (asterisks) in adult rat ovary. Original magnification: 100 (scale bars, 80 lm).
adult group showed a moderate staining for IGF-I (Fig. 6A) and for TGF-b2 (Fig. 6B). However, no staining for bFGF and a weak staining for EGF-R was observed (Fig. 6C and D). 4. Discussion In this study, we intended to detect the expression and localization of IGF-I, TGF-b2, bFGF and EGF-R in rat ovaries of different ages by immunohistochemistry. It is reviewed that IGF-I stimulated proliferation or differentiation of granulosa cells, depending on the stage of development of the follicle and it also stimulates proliferation of granulosa cells from small follicles [16]. In addition it was claimed that IGF family
plays an important role in follicle development, dominant follicle growth, steroidogenesis and follicular atresia [17]. Previous studies reported that IGF-I regulates human luteal steroidogenesis by an increase in progesterone [18] and oestradiol production [19]. IGFI was regarded as a co-gonadotrophin of LH in stimulating steroidogenesis in theca-intertitial cells [20]. In this study we found a moderate staining for IGF-I in granulosa cells of newborn rats, a strong staining in oocytes of young rats, a strong staining in stroma cells of young and adult rats, a moderate staining in theca cells of adult rats and also a moderate staining persisted in the corpus luteii. As it was seen, IGF-1 affected different parts of the ovary follicles in different ages. Hormonal changes with increasing age and development of the follicles might be the cause of these findings.
K. Ergin et al. / Cytokine 43 (2008) 209–214
213
Fig. 6. Corpus luteii in the adult group (A) Moderate staining for IGF-I, (B) Moderate staining for TGF-b2, (C) No staining for bFGF, (D) Weak staining for EGF-R. Original magnification: 200 (scale bars, 40 lm).
One family of peptides known to be involved in the regulation of ovarian development and function is the TGF-b superfamily [11]. Liu et al. were found that TGF-b increases the diameter of follicles from adult mice and has no effect on preantral follicles from immature mice [21]. TGF-b did not have an effect on preantral follicles from 11-day-old mice [21]. On the other hand, TGF-b stimulated the follicular growth of 56-day-old mice [21]. Interestingly, preantral follicles of 28-day-old mice showed an intermediate response, suggesting that the different responses are related to the process of physical maturity [21]. In comparison of newborn, young and adult rats, we observed TGF-b staining only in oocytes of young and adult rats in a variety of degrees. As a result of these findings, we thought that the effect of TGF-b does not appear until puberty. It is reviewed by Knight et al. that members of the TGF-b superfamily, are expressed in oocytes in a developmental-stage related manner and act as intraovarian regulatory molecules [22]. Juengel and McNatty reported that TGF-b2 was found in the oocytes of secondary follicles in rodents [23]. Similarly, we found stronger TGFb2 staining in the oocytes of secondary follicles in young and adult rats. We suggested that TGF-b actions may be age and stage dependent. In the study of Chegini and Flanders, only small luteal cells showed intense immunostaining for TGF-b2; however, the large luteal cells had a weak immunostaining at midluteal phase in human ovary [24]. Besides, ovarian stromal cells do not immunostain for TGF-b2 [24]. In this study, we found a moderate staining of TGF-
b2 in the cells of the corpus luteii and of the stroma. The difference of staining intensity might be due to different sizes of corpus luteii cells. Thus it could be the limitation of our study, not to focus on the sizes of corpus luteii cells and stages of the luteal phase. On the other hand, compared to Chegini and Flanders’ study, the difference of staining intensity in stromal cells might be because of using rat ovaries via human ovaries. In this respect, we could suggest that TGF-b2 may affect the oocyte development and corpus luteum survival. The difference between the species might affect the expression of growth factors such as bFGF. Ben-haroush et al. were detected bFGF in granulosa cells from primary follicular stages onward of adolescents/women, and in oocytes from fetuses and adults at all follicular classes [12]. Besides Nilsson et al. were found that bFGF was localized to primordial and early developing follicles and were mainly observed in the oocytes of 4-day-old rat ovaries which were isolated and cultured for 14 days. In that study, elevated levels of bFGF in the oocytes of primordial and early developing follicles was described [13]. In our study, we found strong immunostaining in oocytes of primary and secondary follicles of young rats, moderate staining in oocytes of secondary and tertiary follicles of adult rats and no staining in newborn rats. Because we used different age groups of rats such as newborn (0-day), young and adults, and studied only immunohistochemically, our findings were not in accordance with Nilsson’s study. In addition to this, our findings
214
K. Ergin et al. / Cytokine 43 (2008) 209–214
showed that bFGF affected follicles especially in young and adult rats. In some studies EGF-receptor (EGF and TGFa bind to this receptor) has been identified in granulosa cells, corpus luteum and oocytes [25,26]. Dark staining was found in the oocytes compared to follicular and stromal cells in human fetuses [25]. Bennet et al. postulated that EGF-R may act as local regulators for the development or degeneration of individual oocytes [25]. We found a strong staining for EGF-R in oocytes of newborn rats, a moderate staining in one-month-old rats and a weak staining in adult rats. From this point of view, a decreased staining of EGF-R was seen by increasing age. Therefore, we thought that the effect of EGF-R were decreasing with age and hence EGF-R might have the strongest effects in newborn term. Ayyagari and Khan-Dawood demonstrated the presence of EGFreceptors in human corpus luteum [26]. We found a weak staining in corpus luteii for EGF-R. Since EGF-R staining decreased with age in oocytes, this was an expected result. In conclusion, we have demonstrated the presence of IGF-I, TGFb2, bFGF and EGF-R proteins in developing ovaries, suggesting that growth factors play a pivotal role in ovarian function. The role of growth factors in controlling degeneration or growth (or both) of ovary follicles remain as explained. Therefore, additional studies should be undertaken to elucidate the role of these and other proteins in development and atresia of ovarian follicles. Acknowledgments The authors thank the Adnan Menderes University Research Fund for financial support (TPF-4015) for this study and Assist. Prof. Dr. Yüksel Yıldız for his language assistance. References [1] Hsueh AJ, Billig H, Tsafriri A. Ovarian follicle atresia: a hormonally controlled apoptotic process. Endocr Rev 1994;15:707–24. [2] Quirk SM, Cowan RG, Harman RM, Hu CL, Porter DA. Ovarian follicular growth and atresia: the relationship between cell proliferation and survival. J Anim Sci 2004;82(E-Suppl):E40–52. [3] Kaiser GG, Sinowatz F, Palma GA. Effects of growth hormone on female reproductive organs. Anat Histol Embryol 2001;30:265–71. [4] Ulug U, Turan E, Tosun SB, Erden HF, Bahceci M. Comparison of preovulatory follicular concentrations of epidermal growth factor, insulin-like growth factor-I, and inhibins A and B in women undergoing assisted conception treatment with gonadotropin-releasing hormone (GnRH) agonists and GnRH antagonists. Fertil Steril 2007;87:995–8. [5] Luciano AM, Modina S, Gandolfi F, Lauria A, Armstrong DT. Effect of cell-to-cell contact on in vitro deoxyribonucleic acid synthesis and apoptosis responses of bovine granulosa cells to insulin-like growth factor-I and epidermal growth factor. Biol Reprod 2000;63:1580–5. [6] Monget P, Fabre S, Mulsant P, Lecerf F, Elsen JM, Mazerbourg S, et al. Regulation of ovarian folliculogenesis by IGF and BMP system in domestic animals. Domest Anim Endocrinol 2002;23:139–54.
[7] Mazerbourg S, Bondy CA, Zhou J, Monget P. The insulin-like growth factor system: a key determinant role in the growth and selection of ovarian follicles? a comparative species study. Reprod Domest Anim 2003;38:247–58. [8] Villavicencio A, Iñiguez G, Johnson MC, Gabler F, Palomino A, Vega M. Regulation of steroid synthesis and apoptosis by insulin-like growth factor I and insulin-like growth factor binding protein 3 in human corpus luteum during the midluteal phase. Reproduction 2002;124(4):501–8. [9] Nikolettos N, Asimakopoulos B, Köster F, Schöpper B, Scultz C, Caglar GS, et al. Cytokine profile in cases with premature elevation of progesterone serum concentrations during ovarian stimulation. Physiol Res 2007;8. [Epub ahead of print]. [10] Monniaux D, Monget P, Besnard N, Huet C, Pisselet C. Growth factors and antral follicular development in domestic ruminants. Theriogenology 1996;47:3–12. [11] Drummond AE. TGF beta signalling in the development of ovarian function. Cell Tissue Res 2005;322(1):107–15. [12] Ben-Haroush A, Abir R, Ao A, Jin S, Kessler-Icekson G, Feldberg D, et al. Expression of basic fibroblast growth factor and its receptors in human ovarian follicles from adults and fetuses. Fertil Steril 2005;849(Suppl 2):1257–68. [13] Nilsson E, Parrott JA, Skinner MK. Basic fibroblast growth factor induces primordial follicle development and initiates folliculogenesis. Mol Cell Endocrinol 2001;175(1–2):123–30. [14] Chen YG, Meng AM. Negative regulation of TGF-beta signaling in development. Cell Res 2004;14(6):441–9. [15] Méndez C, Alcántara L, Escalona R, López-Casillas F, Pedernera E. Transforming growth factor beta inhibits proliferation of somatic cells without influencing germ cell number in the chicken embryonic ovary. Cell Tissue Res 2006;325(1):143–9. [16] Monget P, Fabre S, Mulsant P, Lecerf F, Elsen JM, Mazerbourg S, et al. Regulation of ovarian folliculogenesis by IGF and BMP system in domestic animals. Domest Anim Endocrinol 2002;23(1–2):139–54. [17] Giudice LC. Insulin-like growth factor family in Graafian follicle development and function. J Soc Gynecol Investig 2001;8:S26–9. [18] Devoto L, Kohen P, Castro O, Vega M, Troncoso JL, Charreau E. Multihormonal regulation of progesterone synthesis in cultured human midluteal cells. J Clin Endocrinol Metab 1995;80(5):1566–70. [19] Johnson MC, Devoto L, Retamales I, Kohen P, Troncoso JL, Aguilera G. Localization of insulin-like growth factor (IGF-I) and IGF-I receptor expression in human corpora lutea: role on estradiol secretion. Fertil Steril 1996;65(3):489–94. [20] Duleba AJ, Spaczynski RZ, Olive DL, Behrman HR. Divergent mechanisms regulate proliferation/survival and steroidogenesis of theca-interstitial cells. Mol Hum Reprod 1999;5(3):193–8. [21] Liu X, Andoh K, Abe Y, Kobayashi J, Yamada K, Mizunuma H, et al. A comparative study on transforming growth factor-beta and activin A for preantral follicles from adult, immature, and diethylstilbestrol-primed immature mice. Endocrinology 1999;140(6):2480–5. [22] Knight PG, Glister C. Local roles of TGF-beta superfamily members in the control of ovarian follicle development. Anim Reprod Sci 2003;78(3–4): 165–183. [23] Juengel JL, McNatty KP. The role of proteins of the transforming growth factorbeta superfamily in the intraovarian regulation of follicular development. Hum Reprod Update 2005;11(2):143–60. [Epub 2005 Feb 10]. [24] Chegini N, Flanders KC. Presence of transforming growth factor-beta and their selective cellular localization in human ovarian tissue of various reproductive stages. Endocrinology 1992;130(3):1707–15. [25] Bennett RA, Osathanondh R, Yeh J. Immunohistochemical localization of transforming growth factor-alpha, epidermal growth factor (EGF), and EGF receptor in the human fetal ovary. J Clin Endocrinol Metab 1996;81(8):3073–6. [26] Ayyagari RR, Khan-Dawood FS. Human corpus luteum: presence of epidermal growth factor receptors and binding characteristics. Am J Obstet Gynecol 1987;156(4):942–6.