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Fibroblast growth factor 10 in human ovaries
Reproductive BioMedicine Online (2012) 25, 396– 401
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ARTICLE
Fibroblast growth factor 10 in human ovaries Galia Oron a,b, Benjamin Fisch a,b, Xiao Yun Zhang c, Rinat Gabbay-Benziv a,b, Gania Kessler-Icekson d,b, Haim Krissi e, Avi Ben-Haroush a,b, Asangla Ao c,f, Ronit Abir a,b,* a Infertility and IVF Unit, Hospital for Women, Rabin Medical Center, Petach Tikva 49100, Israel; b Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; c Department of Obstetrics and Gynecology, Royal Victoria Hospital, McGill University, Montreal, Quebec, Canada; d The Felsenstein Medical Research Center, Beilinson Hospital, Petah Tikva 49100, Israel; e Beilinson Women’s Hospital, Rabin Medical Center, Petach Tikva, Israel; f Department of Human Genetics, McGill University, Montreal, Quebec, Canada
* Corresponding author. E-mail address: [email protected] (R Abir). Dr Oron completed her residency and is a senior physician in obstetrics and gynaecology at the Hospital for Women, Rabin Medical Center, Israel. She is currently serving as a lecturer at Tel-Aviv University, Israel, from which she received the best student tutor award in 2011. Her study ‘Pregnancy outcome in women with heart disease undergoing induction of labor’ is cited in the 22nd edition of Williams Obstetrics as reference guidance. She has recently published two articles investigating the expression of growth differentiation factor 9 and neurotrophin 3 and their effect the growth and maturation of early human ovarian follicles.
Abstract The expression of fibroblast growth factor 10 (FGF-10) has not been studied in human ovarian cortical follicles. The aim of
the present study was to investigate the expression of FGF-10 in preantral follicles from fetuses, girls and women. Ovarian samples were obtained from 14 human fetuses at 21–33 gestational weeks and from 35 girls and women aged 5–39 years. The specimens were prepared for detection of the FGF-10 protein by immunohistochemistry. Reverse-transcription PCR was applied to ovarian extracts to identify FGF-10 mRNA transcripts. In fetal tissue, the FGF-10 protein was detected in oocytes in 50% of the samples and in granulosa cells in 30%. In ovarian tissue from girls and women, the FGF-10 protein was detected in oocytes and granulosa cells in all samples. FGF-10 mRNA transcripts were present in all adult and fetal samples tested. The identification of FGF-10 at both the protein and mRNA levels suggests that FGF-10 may contribute to human preantral follicle development. RBMOnline ª 2012, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: fibroblast growth factor 10 (FGF-10), granulosa cells, human preantral follicles, immunohistochemistry, primordial/preantral follicles, reverse-transcription PCR
Introduction Improvements in anticancer treatment have resulted in a significant increase in survival of young female patients (Abir et al., 2006, 2008; Feigin et al., 2008). One of the side
effects of the anticancer therapy is follicular depletion and infertility. Very early ovarian follicles are the lifetime fertility reserve in women. Thus, a major option for preserving fertility in patients with cancer is the cryopreservation of primordial-follicle-containing ovarian tissue before
1472-6483/$ - see front matter ª 2012, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.rbmo.2012.07.002
Fibroblast growth factor 10 in human ovaries initiation of therapy, followed by reimplantation of the frozen–thawed ovarian tissue (Dittrich et al., 2012; Silber, 2012). However, to eliminate the risk of reseeding cancer using this procedure (Abir et al., 2006, 2008), a successful system for growing primordial follicles in culture is necessary. This requires in-depth study of the still-unknown signals that are responsible for triggering the development of primordial follicles (Abir et al., 2006; Telfer et al., 2008). Fibroblast growth factors (FGF) constitute one of the largest families (22 members) of growth and differentiation factors in cells of mesodermal and neuroectodermal origin (Ornitz and Itoh, 2001). They have been implicated in a variety of cellular processes, including angiogenesis, cell survival, cell migration and chemotaxis (Basilico and Moscatelli, 1992; Bottcher and Niehrs, 2005). Fibroblast growth factor 10 (FGF-10), also known as keratinocyte growth factor 2 (KGF2), is a 13.9 kDa heparin-binding protein involved in cell migration, epithelial cell motility, cell differentiation and wound healing (Ware and Matthay, 2002). It shares the same unique receptor with KGF (FGF-7), the KGF receptor (KGFR) also termed FGFR2b or FGFR2-IIIb, a splicing variant involving alternative exons of the FGF receptor 2 gene (FGFR2; Beenken et al., 2012; de Giorgi et al., 2007; Finch and Rubin, 2004; Rubin et al., 1995; Steinberg et al., 2004; Wilkie et al., 2002). The extracellular domain of both KGFR and FGFR2 contains three immunoglobulin (Ig)-like loops, and their only sequence difference lies in the C-terminal closest to the intramembranal part (receptor stem), where amino acid similarity is only 47%. KGFR is formed in two Ig-like loops (IgIII domains) with the alternatively spliced IgIIIa/IIIb exons. FGF take part in ovarian folliculogenesis, including the regulation of preantral (primordial to secondary) development (Abir et al., 2009; Ben-Haroush et al., 2005; Buratini et al., 2005, 2007; Machado et al., 2009). In the two reports on higher mammals to date (Buratini et al., 2007; Chaves et al., 2010), FGF-10 protein and mRNA transcripts were identified in oocytes of bovine preantral follicles (Buratini et al., 2007), and in-vitro culture of preantral follicles from goats with FGF-10 maintained follicular morphological integrity and stimulated primordial follicular growth (Chaves et al., 2010). The expression of mRNA transcripts for KGFR have previously been identified in human ovaries from fetuses, girls and women and also in the oocytes and granulosa cells (Abir et al., 2009). The aim of the present study was to investigate, as far as is known for the first time, if the FGF-10 protein (by immunohistochemistry, IHC) and its mRNA transcripts (by reverse-transcription PCR, RT-PCR) are expressed in human ovaries from fetuses, girls and women.
Materials and methods Human ovaries from fetuses, girls and women Ovarian samples were obtained from 14 aborted human fetuses aged 21–33 gestational weeks. In addition, small ovarian biopsy samples were donated by 35 girls and women aged 5–39 years or their parents. All had undergone gynaecological laparoscopies for either cryopreservation of ovarian tissue before commencement of chemotherapy or
397 removal of cysts (Abir et al., 2009). The Ethics Committee of Rabin Medical Centre approved the study protocol (certificate number 5872, approved 6 June 2010), and every woman or minor’s parents signed an informed consent form. The samples were cut into uniform size and fixed immediately for IHC studies (Abir et al., 2009; Pinkas et al., 2008). Samples were then frozen for subsequent RNA extraction.
Cryopreservation of ovarian tissue Tissue slices were placed in cryogenic vials (Nalge Nunc International, Roskilde, Denmark) filled with a solution of 1.5 mol/l dimethylsulphoxide (Sigma, St Louis, MO, USA; Abir et al., 2009; Pinkas et al., 2008). Prior to freezing, the samples were kept on ice for 30 min to achieve equilibrium. All samples were frozen in a programmable freezer (Kryo 360-1/7, Planer Biomed, Sunbury on Thames, UK) and immediately placed in liquid nitrogen. The slices were cryopreserved-stored for up to 5 years until RNA extraction.
Histological preparation The histological preparation method has been described in detail elsewhere (Abir et al., 2009; Pinkas et al., 2008). The fixed specimens were dehydrated in a graded series of ethanol followed by paraffin embedding and sectioning. Unstained sections were placed on OptiPlus positive-charged microscope slides (BioGenex Laboratories, San Ramon, CA, USA) for IHC.
IHC for FGF-10 The IHC method has been employed in several earlier studies (Abir et al., 2009; Ben-Haroush et al., 2005; Pinkas et al., 2008). Two sections per sample were utilized to identify the protein for FGF-10. To enhance antigen retrieval, all the slides were microwaved with citrate buffer at pH 6.0 (CheMate buffer; DAKOCytomation, Glostrup, Denmark), and to block endogenous peroxidase activity, the slides were quenched in 3% hydrogen peroxide (Vitamed, Binyamina, Israel). Unfortunately, there were no commercially available positive controls for FGF-10. The primary antibody was goat polyclonal antibody against FGF-10 (sc-sc27147; Santa Cruz Biotechnology), a specific antibody recommended by the suppliers for immunostaining. The samples were incubated with the goat polyclonal antibody against FGF-10 diluted 1/10 and 1/30. Negative control solutions were prepared by absorption of this diluted primary antibody with its corresponding blocking peptide (sc27147P; Santa Cruz Biotechnology). Thereafter, the samples were incubated with horseradish peroxide polymer conjugated anti-goat secondary antibodies (SuperPicture HRP, 879363; Zymed Laboratories, San Francisco, CA, USA). Red-brown 3-amino-9-ethylcarbazole (Zymed Laboratories) staining indicated the presence of the antigen, with blue Mayer’s haematoxylin (Pioneer Research Chemicals, Colchester Essex, UK) counterstaining.
Follicular counts The number of follicles in every IHC-stained section was counted with an image analyser (analySIS; Soft Imaging
398
G Oron et al.
System Munster, Germany) and the follicles were classified according to Gougeon (1996): primordial = with a single flat layer of ovarian granulosa cells (GC) surrounding the oocyte (the majority of follicles in mammalian ovaries); primary = with cuboidal GC (following activation of primordial follicles); secondary = with multiple layers of GC surrounding the oocyte and with a theca cell layer; and antral = with a fluid-filled cavity within the GC (the final development stage).
Reverse-transcription PCR
staining (i.e., blue staining) was considered a negative staining. Figure 1A shows the characteristic cellular localization of the FGF-10 protein in ovarian tissue from fetuses. Full positive cytoplasmic staining was identified in 50% of the samples without nuclear staining. The GC were weakly stained in 30% of the fetal samples. Figure 1B shows the characteristic cellular localization of the FGF-10 protein in ovarian tissue from girls and women. Full positive cytoplasmic staining was noted in all samples with nuclear staining. The GC were weakly stained in all samples. The negative controls for both fetuses and girls/women stained negatively (blue) (Figure 1C and D). A portion of the stroma cells in all the samples expressed the protein for FGF-10. There was no further association of the results with ovarian source (fetuses, girls and women), fetal abnormalities, age or follicular class.
The protocol for mRNA extraction from frozen ovarian fragments has been described in detail elsewhere (Abir et al., 2009; Ben-Haroush et al., 2005). RT-PCR was performed essentially as described previously. A single-round PCR approach was used to detect transcripts of Homo sapiens FGF-10 (accession number NM_004465). Primers for exon 1 and exon 2 were used, as published by Ropiquet et al. (2000). The annealing temperature was 57C and the expected PCR product size was 308 bp. The b-actin gene served as a positive control (274 bp; Abir et al., 2010). PCR products were separated on 2% agarose gel along with a 100-bp DNA ladder as a fragment-size reference and were stained with ethidium bromide.
Follicular counts
Results
FGF-10 gene expression
IHC detection of FGF-10 protein
Figure 2A depicts a representative RT-PCR gel showing FGF-10 gene expression in the fetal and adult ovaries. The transcripts for FGF-10 were present in all adult and fetal samples tested. Findings for the control b-actin gene were positive in all samples tested (Figure 2B). None of the negative controls processed without RT yielded an amplification product (Figure 2A and B, lane 9).
Table 1 summarizes the IHC findings for the expression of the FGF-10 protein in ovarian samples from fetuses, girls and women. Positive red-brown staining was detected in all the samples tested. In the oocyte, findings were subclassified into nuclear staining and cytoplasmic staining. Positive oocyte staining was described as full (staining in the whole cytoplasm) or partial. An absence of red-brown
Table 1 Fibroblast growth factor 10 protein expression by immunohistochemistry in human ovaries. Source
Level of expression
Fetus Oocyte Cytoplasm Nucleus GC Stroma Girls/women Oocyte Cytoplasm Nucleus GC Stroma
Weak 50% + + (full) 30% + + Weak–medium + + (full) + Weak +
GC = granulosa cells; – = no staining; + = staining; full = full staining.
In the IHC samples from fetuses, 1572 follicles were counted: 1445 (92%) primordial, 126 (8%) primary and one (0.1%) secondary. In the IHC samples from girls and women, 871 follicles were counted: 706 (81%) primordial, 153 (18%) primary and 12 (1%) secondary.
Discussion As far as is known, this is the first study to provide information on the presence of FGF-10 in human preantral follicles from fetuses, girls and women. The IHC results revealed the expression of FGF-10 in oocytes in 50% of the fetal samples (without nuclear staining) and weak GC staining in 30%. In girls and women, positive staining was detected in the oocyte (with nuclear staining) and in the GC in all samples. From RT-PCR, FGF-10 mRNA transcripts were present in all specimens from fetuses and women. Age-related developmental changes can explain the stronger expression and wider distribution of the FGF-10 protein in the samples from girls and women than in samples from fetuses, as well as the identification of oocyte nuclear staining only in the samples from girls and women. Lack of oocyte nuclear staining in samples from fetuses is in line with similar results in previous studies on expression absence of other growth factors and their receptors in human fetal oocyte nuclei (Abir et al., 2010; Ben-Haroush et al., 2005; Pinkas et al., 2008). In general, the present study found a good correspondence between FGF-10 protein and mRNA expression. However, protein staining was weak in the fetal samples, whereas the corresponding mRNA transcripts were easily identified by RT-PCR. This finding might
Fibroblast growth factor 10 in human ovaries
399
Figure 1 Immunohistochemistry micrographs of fibroblast growth factor 10 (FGF-10) protein expression. (A) Section of human ovary from a 27-gestational-week-old fetus. Note the primordial follicles, the red-brown staining indicating FGF-10 in oocytes (full cytoplasmic but not nuclear staining) with staining in the GC and staining in a portion of the stroma cells. (B) Section of human ovary from a 13-year-old girl. Note the secondary follicle with the red-brown staining indicating FGF-10 protein in the oocyte (full cytoplasmic and nuclear staining), and in the GC and a portion of the stroma cells. (C) Negative control for the same ovarian section as A: note the primordial follicles with the overall blue staining and the lack of red-brown staining. (D) Negative control for the same ovarian section as B: note the primordial follicles with the overall blue staining and the lack of red-brown staining. Bars = 30 lm.
Figure 2 Representative reverse-transcription PCR gel illustrating fibroblast growth factor 10 (FGF-10) gene expression in fetal and adult ovaries: (A) FGF-10 (308 bp); (B) b-actin control (274 bp). Lanes: 1 = 100 bp DNA ladder; 2 = 21-year-old woman; 3 = 29-year-old woman; 4 = 39-year-old woman; 5 = 21-gestational-week-old fetus; 6 = 22-gestational-week-old fetus; 7 = 25-gestational-week-old fetus; 8 = 27-gestational-week-old fetus; 9 = negative PCR control.
be attributable to a higher RT-PCR sensitivity to low levels of mRNA transcripts relative to the IHC sensitivity to the relevant protein. Be that as it may, the RT-PCR assay was qualitative and not quantitative and, therefore, could not detect changes in mRNA levels. There is very limited information regarding the connection between FGF-10 and mammalian preantral follicles. The present results are in line with a study in cows, wherein
the FGF-10 protein was detected in oocytes of preantral follicles and FGF-10 mRNA transcripts were detected in oocytes at all developmental stages and also in theca cells (Buratini et al., 2007). The differences in the cellular staining pattern between the bovine (Buratini et al., 2007) and the human (the present study) can be attributed to species differences. Accordingly, Chaves et al. (2010) reported that follicular morphology was maintained, with stimulation of
400 follicular growth from the primordial to primary stage, following in-vitro culture of goat preantral follicles with FGF-10. In an earlier work by this study group, mRNA transcripts for the FGF-10 receptor, KGFR, were identified in human ovaries from fetuses, girls and women by in-situ hybridization (Abir et al., 2009). Five of the fetal samples from that study and six of the samples from girls and women were also tested in the present study for the FGF-10 protein. However, follicular KGFR mRNA was found in only 10% of the original samples (Abir et al., 2009), whereas its FGF-10 protein ligand was identified in a larger proportion of samples. This discrepancy may be explained by the higher sensitivity of IHC to protein than of in-situ hybridization for mRNA transcripts in paraffin sections (Pinkas et al., 2008). Additionally, it is noteworthy that although no specific antibody against KGFR is commercially available, the amino acid sequence within the C-terminal cytoplasmic domain is identical in KGFR and FGFR2 (Abir et al., 2009; de Giorgi et al., 2007; Finch and Rubin, 2004; Rubin et al., 1995; Wilkie et al., 2002). Using an antibody targeted against a peptide mapped within this domain, the present study group has identified the FGFR2 protein in oocytes of preantral follicles from human fetuses and girls/women and in GC of girls/women (Ben-Haroush et al., 2005). Therefore, it is likely that the immunostaining procedure for FGFR2 also picked up the KGFR protein (Abir et al., 2009; Ben-Haroush et al., 2005). This is the group’s third report of the identification of FGF members that might be connected with early folliculogenesis in humans, in particular activation of primordial follicles (Abir et al., 2009; Ben-Haroush et al., 2005). The expression of FGF-2 (basic FGF) and FGF-7 and their receptors in human ovaries have been previously identified in fetuses, girls and women (Abir et al., 2009; Ben-Haroush et al., 2005). This group has also shown that FGF-2 promoted 17-b-oestradiol production in cultured human follicles, and contributed marginally to the activation of human primordial follicles (Garor et al., 2008). The present and previous findings (Abir et al., 2009; Ben-Haroush et al., 2005; Garor et al., 2008) shed light on the contribution of the FGF system to the complex interplay between various growth factors in the initial follicular developmental stages in humans, as also reported for other mammals (Buratini et al., 2005). Moreover, this is the third report connecting FGF-10 and preantral follicles in higher mammals (Buratini et al., 2007; Chaves et al., 2010). Be that as it may, definite conclusions regarding the possible role of FGF-10 in growth promotion of human primordial follicles can be drawn only after FGF-10 is included in their culture medium. Additional studies in humans are needed to clarify the role of FGF-10, other growth factors and their synergistic interactions in preantral follicles.
Acknowledgements The authors are greatly indebted to their laboratory technician, Carmela Felz, for preparation of the histological sections. They are grateful to Gloria Ganzach from the Editorial Board, Beilinson Hospital, Rabin Medical Centre for the English editing, to the staff at the gynaecology ward
G Oron et al. for their help in locating suitable patients and to the Ultrasound Unit for identifying fetal gender (all from Beilinson Hospital, Rabin Medical Centre).
References Abir, R., Nitke, S., Ben-Haroush, A., Fisch, B., 2006. In vitro maturation of human primordial ovarian follicles – clinical significance, progress and methods for growth evaluation. Histol. Histopathol. 21, 887–898. Abir, R., Ben-Haroush, A., Felz, C., Okon, E., Raanani, H., Orvieto, R., et al., 2008. Selection of patients before and after anticancer treatment for ovarian cryopreservation. Hum. Reprod. 23, 869–877. Abir, R., Fisch, B., Zhang, X.Y., Felz, C., Kessler-Icekson, G., Krissi, H., Nitke, S., Ao, A., 2009. Keratinocyte growth factor and its receptor in human ovaries from fetuses, girls and women. Mol. Hum. Reprod. 15, 69–75. Abir, R., Ao, A., Zhang, X.Y., Garor, R., Nitke, S., Fisch, B., 2010. Vascular endothelial growth factor A and its two receptors in human preantral follicles from fetuses, girls, and women. Fertil. Steril. 93, 2337–2347. Basilico, C., Moscatelli, D., 1992. The FGF family of growth factors and oncogenes. Adv. Cancer. Res. 59, 115–165. Beenken, A., Eliseenkova, A.V., Ibrahimi, O.A., Olsen, S.K., Mohammadi, M., 2012. Plasticity in interaction of fibroblast growth factor 1 (FGF1) N terminus with FGF receptors underlines promiscuity of FGF1. J. Biol. Chem. 287, 3067–3078. Ben-Haroush, A., Abir, R., Ao, A., Jin, S., Kessler-Icekson, G., Fldberg, D., Fisch, B., 2005. Expression of basic fibroblast growth factor and its receptors in human ovarian follicles from adults and fetuses. Fertil. Steril. 84, 1257–1268. Bottcher, R.T., Niehrs, C., 2005. Fibroblast growth factor signaling during early vertebrate development. Endoc. Rev. 26, 63–77. Buratini, J., Teixeira, A.B., Costa, I.B., Glapinski, V.F., Pinto, M.G.L., Giometti, I.C., Baros, C.M., 2005. Expression of fibroblast growth factor-8 and regulation of cognate receptors, fibroblast growth receptor-3c and-4 in bovine antral follicles. Reproduction 130, 343–350. Buratini, J., Pinto, M.G.L., Castilho, A.C., Amorim, R.L., Giometti, I.C., Portela, V.M., Nicola, E.S., 2007. Expression and function of fibroblast growth factor 10 and its receptor, fibroblast growth factor receptor 2B in bovine follicles. Biol. Reprod. 77, 743–750. Chaves, R.N., Lima-Verde, I.B., Celestino, J.J.H., Duarte, A.B.G., Alves, A.M.C.V., Matos, M.H.T., Campello, C.C., Name, K.P., Bao, S.N., Buratini Jr., J., Figueiredo, J.R., 2010. Fibroblast growth factor-10 maintains the survival and promotes the growth of cultured goat preantral follicles. Domestic Animal Endocrinol. 39, 249–258. De Giorgi, V., Sestini, S., Massi, D., Ghersetich, I., Torello, L., 2007. Keratinocyte growth factor receptors. Dermatol. Clin. 25, 477–485. Dittrich, R., Lotz, L., Keck, G., Hoffmann, I., Mueller, A., Beckmann, M.W., van den Ven, H., Montag, M., 2012. Live birth after ovarian tissue autotransplantation following overnight transportation before cryopreservation. Fertil. Steril. 97, 387–390. Feigin, E., Freud, E., Fisch, B., Orvieto, R., Kravarusic, D., Avrahami, G., Ben-Haroush, A., Abir, R., 2008. Fertility preservation in female adolescents with malignancies. In: Cancer in Female Adolescents. USA Science Publishers Inc., Hauppauge, NY, pp. 38–101. Finch, P.W., Rubin, J.S., 2004. Keratinocyte growth factor/fibroblast growth factor 7, a homeostatic factor with therapeutic potential for epithelial protection and repair. Adv. Cancer Res. 91, 69–136.
Fibroblast growth factor 10 in human ovaries Garor, R., Abir, R., Erman, A., Felz, C., Nitke, S., Fisch, B., 2008. The in vitro effects of basic fibroblast growth factor on human primordial follicles. Fertil. Steril. 91, 1967–1975. Gougeon, A., 1996. Regulations of ovarian follicular development in primates: facts and hypotheses. Endocrinol. Rev. 17, 121–154. Machado, M.F., Portela, M.F., Price, C.A., Costa, I.B., Ripamonte, P., Amorim, R.L., Buratini Jr., J., 2009. Regulation and action of fibroblast growth factor 17 in bovine follicles. J. Endocrinol. 202, 347–353. Ornitz, D.M., Itoh, N., 2001. Fibroblast growth factors. Genome. Biol. 2, 1–12. Pinkas, H., Fisch, B., Rozansky, G., Felz, C., Kessler-Icekson, G., Krissi, H., Nitke, S., Ao, A., Abir, R., 2008. Platelet-derived growth factors (PDGF-A and -B) and their receptors in human fetal and adult ovaries. Mol. Hum. Reprod. 14, 199–206. Ropiquet, F., Giri, D., Kwabi-Addo, B., Schmidt, K., Ittmann, M., 2000. FGF-10 is expressed at low levels in the human prostate. Prostate 44, 334–338. Rubin, J., Bottaro, D., Chedid, M., Miki, T., Ron, D., Cheon, G., Taylor, W.G., Fortney, E., Sakata, H., Finch, P.W., LaRochelle, W.J., 1995. Keratonocyte growth factor. Cell. Biol. Int. 19, 399–407. Silber, S.J., 2012. Ovary cryopreservation and transplantation for fertility preservation. Mol. Hum. Reprod. 18, 59–67.
401 Steinberg, Z., Myers, C., Heim, V.M., Lathrop, C.A., Rubustini, I.T., Stewart, J.S., Larsen, M., Hoffman, M.P., 2004. FGFR2b signaling regulates ex vivo submandibular gland epithelial cell proliferation and branching morphogenesis. Development 132, 1223–1234. Telfer, E.E., McLaughlin, M., Ding, C., Thong, K.J., 2008. A two-step serum-free culture system supports development of human oocytes from primordial follicles in the presence of activin. Hum. Reprod. 23, 1151–1158. Ware, L.B., Matthay, M.A., 2002. Keratinocyte and hepatocyte growth factors in the lung: roles in lung development, inflammation and repair. Am. J. Physiol. 282, 924–940. Wilkie, A.O.M., Patey, S.J., Kan, S., van den Ouweland, A.M.W., Hamel, B.C.J., 2002. FGFs, their receptors, and human limb malformations: clinical and molecular correlations. Am. J. Med. Gen. 112, 266–278. Declaration: The authors report no financial or commercial conflicts of interest. Received 11 March 2012; refereed 12 June 2012; accepted 11 July 2012.
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ARTICLE
Fibroblast growth factor 10 in human ovaries Galia Oron a,b, Benjamin Fisch a,b, Xiao Yun Zhang c, Rinat Gabbay-Benziv a,b, Gania Kessler-Icekson d,b, Haim Krissi e, Avi Ben-Haroush a,b, Asangla Ao c,f, Ronit Abir a,b,* a Infertility and IVF Unit, Hospital for Women, Rabin Medical Center, Petach Tikva 49100, Israel; b Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel; c Department of Obstetrics and Gynecology, Royal Victoria Hospital, McGill University, Montreal, Quebec, Canada; d The Felsenstein Medical Research Center, Beilinson Hospital, Petah Tikva 49100, Israel; e Beilinson Women’s Hospital, Rabin Medical Center, Petach Tikva, Israel; f Department of Human Genetics, McGill University, Montreal, Quebec, Canada
* Corresponding author. E-mail address: [email protected] (R Abir). Dr Oron completed her residency and is a senior physician in obstetrics and gynaecology at the Hospital for Women, Rabin Medical Center, Israel. She is currently serving as a lecturer at Tel-Aviv University, Israel, from which she received the best student tutor award in 2011. Her study ‘Pregnancy outcome in women with heart disease undergoing induction of labor’ is cited in the 22nd edition of Williams Obstetrics as reference guidance. She has recently published two articles investigating the expression of growth differentiation factor 9 and neurotrophin 3 and their effect the growth and maturation of early human ovarian follicles.
Abstract The expression of fibroblast growth factor 10 (FGF-10) has not been studied in human ovarian cortical follicles. The aim of
the present study was to investigate the expression of FGF-10 in preantral follicles from fetuses, girls and women. Ovarian samples were obtained from 14 human fetuses at 21–33 gestational weeks and from 35 girls and women aged 5–39 years. The specimens were prepared for detection of the FGF-10 protein by immunohistochemistry. Reverse-transcription PCR was applied to ovarian extracts to identify FGF-10 mRNA transcripts. In fetal tissue, the FGF-10 protein was detected in oocytes in 50% of the samples and in granulosa cells in 30%. In ovarian tissue from girls and women, the FGF-10 protein was detected in oocytes and granulosa cells in all samples. FGF-10 mRNA transcripts were present in all adult and fetal samples tested. The identification of FGF-10 at both the protein and mRNA levels suggests that FGF-10 may contribute to human preantral follicle development. RBMOnline ª 2012, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. KEYWORDS: fibroblast growth factor 10 (FGF-10), granulosa cells, human preantral follicles, immunohistochemistry, primordial/preantral follicles, reverse-transcription PCR
Introduction Improvements in anticancer treatment have resulted in a significant increase in survival of young female patients (Abir et al., 2006, 2008; Feigin et al., 2008). One of the side
effects of the anticancer therapy is follicular depletion and infertility. Very early ovarian follicles are the lifetime fertility reserve in women. Thus, a major option for preserving fertility in patients with cancer is the cryopreservation of primordial-follicle-containing ovarian tissue before
1472-6483/$ - see front matter ª 2012, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.rbmo.2012.07.002
Fibroblast growth factor 10 in human ovaries initiation of therapy, followed by reimplantation of the frozen–thawed ovarian tissue (Dittrich et al., 2012; Silber, 2012). However, to eliminate the risk of reseeding cancer using this procedure (Abir et al., 2006, 2008), a successful system for growing primordial follicles in culture is necessary. This requires in-depth study of the still-unknown signals that are responsible for triggering the development of primordial follicles (Abir et al., 2006; Telfer et al., 2008). Fibroblast growth factors (FGF) constitute one of the largest families (22 members) of growth and differentiation factors in cells of mesodermal and neuroectodermal origin (Ornitz and Itoh, 2001). They have been implicated in a variety of cellular processes, including angiogenesis, cell survival, cell migration and chemotaxis (Basilico and Moscatelli, 1992; Bottcher and Niehrs, 2005). Fibroblast growth factor 10 (FGF-10), also known as keratinocyte growth factor 2 (KGF2), is a 13.9 kDa heparin-binding protein involved in cell migration, epithelial cell motility, cell differentiation and wound healing (Ware and Matthay, 2002). It shares the same unique receptor with KGF (FGF-7), the KGF receptor (KGFR) also termed FGFR2b or FGFR2-IIIb, a splicing variant involving alternative exons of the FGF receptor 2 gene (FGFR2; Beenken et al., 2012; de Giorgi et al., 2007; Finch and Rubin, 2004; Rubin et al., 1995; Steinberg et al., 2004; Wilkie et al., 2002). The extracellular domain of both KGFR and FGFR2 contains three immunoglobulin (Ig)-like loops, and their only sequence difference lies in the C-terminal closest to the intramembranal part (receptor stem), where amino acid similarity is only 47%. KGFR is formed in two Ig-like loops (IgIII domains) with the alternatively spliced IgIIIa/IIIb exons. FGF take part in ovarian folliculogenesis, including the regulation of preantral (primordial to secondary) development (Abir et al., 2009; Ben-Haroush et al., 2005; Buratini et al., 2005, 2007; Machado et al., 2009). In the two reports on higher mammals to date (Buratini et al., 2007; Chaves et al., 2010), FGF-10 protein and mRNA transcripts were identified in oocytes of bovine preantral follicles (Buratini et al., 2007), and in-vitro culture of preantral follicles from goats with FGF-10 maintained follicular morphological integrity and stimulated primordial follicular growth (Chaves et al., 2010). The expression of mRNA transcripts for KGFR have previously been identified in human ovaries from fetuses, girls and women and also in the oocytes and granulosa cells (Abir et al., 2009). The aim of the present study was to investigate, as far as is known for the first time, if the FGF-10 protein (by immunohistochemistry, IHC) and its mRNA transcripts (by reverse-transcription PCR, RT-PCR) are expressed in human ovaries from fetuses, girls and women.
Materials and methods Human ovaries from fetuses, girls and women Ovarian samples were obtained from 14 aborted human fetuses aged 21–33 gestational weeks. In addition, small ovarian biopsy samples were donated by 35 girls and women aged 5–39 years or their parents. All had undergone gynaecological laparoscopies for either cryopreservation of ovarian tissue before commencement of chemotherapy or
397 removal of cysts (Abir et al., 2009). The Ethics Committee of Rabin Medical Centre approved the study protocol (certificate number 5872, approved 6 June 2010), and every woman or minor’s parents signed an informed consent form. The samples were cut into uniform size and fixed immediately for IHC studies (Abir et al., 2009; Pinkas et al., 2008). Samples were then frozen for subsequent RNA extraction.
Cryopreservation of ovarian tissue Tissue slices were placed in cryogenic vials (Nalge Nunc International, Roskilde, Denmark) filled with a solution of 1.5 mol/l dimethylsulphoxide (Sigma, St Louis, MO, USA; Abir et al., 2009; Pinkas et al., 2008). Prior to freezing, the samples were kept on ice for 30 min to achieve equilibrium. All samples were frozen in a programmable freezer (Kryo 360-1/7, Planer Biomed, Sunbury on Thames, UK) and immediately placed in liquid nitrogen. The slices were cryopreserved-stored for up to 5 years until RNA extraction.
Histological preparation The histological preparation method has been described in detail elsewhere (Abir et al., 2009; Pinkas et al., 2008). The fixed specimens were dehydrated in a graded series of ethanol followed by paraffin embedding and sectioning. Unstained sections were placed on OptiPlus positive-charged microscope slides (BioGenex Laboratories, San Ramon, CA, USA) for IHC.
IHC for FGF-10 The IHC method has been employed in several earlier studies (Abir et al., 2009; Ben-Haroush et al., 2005; Pinkas et al., 2008). Two sections per sample were utilized to identify the protein for FGF-10. To enhance antigen retrieval, all the slides were microwaved with citrate buffer at pH 6.0 (CheMate buffer; DAKOCytomation, Glostrup, Denmark), and to block endogenous peroxidase activity, the slides were quenched in 3% hydrogen peroxide (Vitamed, Binyamina, Israel). Unfortunately, there were no commercially available positive controls for FGF-10. The primary antibody was goat polyclonal antibody against FGF-10 (sc-sc27147; Santa Cruz Biotechnology), a specific antibody recommended by the suppliers for immunostaining. The samples were incubated with the goat polyclonal antibody against FGF-10 diluted 1/10 and 1/30. Negative control solutions were prepared by absorption of this diluted primary antibody with its corresponding blocking peptide (sc27147P; Santa Cruz Biotechnology). Thereafter, the samples were incubated with horseradish peroxide polymer conjugated anti-goat secondary antibodies (SuperPicture HRP, 879363; Zymed Laboratories, San Francisco, CA, USA). Red-brown 3-amino-9-ethylcarbazole (Zymed Laboratories) staining indicated the presence of the antigen, with blue Mayer’s haematoxylin (Pioneer Research Chemicals, Colchester Essex, UK) counterstaining.
Follicular counts The number of follicles in every IHC-stained section was counted with an image analyser (analySIS; Soft Imaging
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System Munster, Germany) and the follicles were classified according to Gougeon (1996): primordial = with a single flat layer of ovarian granulosa cells (GC) surrounding the oocyte (the majority of follicles in mammalian ovaries); primary = with cuboidal GC (following activation of primordial follicles); secondary = with multiple layers of GC surrounding the oocyte and with a theca cell layer; and antral = with a fluid-filled cavity within the GC (the final development stage).
Reverse-transcription PCR
staining (i.e., blue staining) was considered a negative staining. Figure 1A shows the characteristic cellular localization of the FGF-10 protein in ovarian tissue from fetuses. Full positive cytoplasmic staining was identified in 50% of the samples without nuclear staining. The GC were weakly stained in 30% of the fetal samples. Figure 1B shows the characteristic cellular localization of the FGF-10 protein in ovarian tissue from girls and women. Full positive cytoplasmic staining was noted in all samples with nuclear staining. The GC were weakly stained in all samples. The negative controls for both fetuses and girls/women stained negatively (blue) (Figure 1C and D). A portion of the stroma cells in all the samples expressed the protein for FGF-10. There was no further association of the results with ovarian source (fetuses, girls and women), fetal abnormalities, age or follicular class.
The protocol for mRNA extraction from frozen ovarian fragments has been described in detail elsewhere (Abir et al., 2009; Ben-Haroush et al., 2005). RT-PCR was performed essentially as described previously. A single-round PCR approach was used to detect transcripts of Homo sapiens FGF-10 (accession number NM_004465). Primers for exon 1 and exon 2 were used, as published by Ropiquet et al. (2000). The annealing temperature was 57C and the expected PCR product size was 308 bp. The b-actin gene served as a positive control (274 bp; Abir et al., 2010). PCR products were separated on 2% agarose gel along with a 100-bp DNA ladder as a fragment-size reference and were stained with ethidium bromide.
Follicular counts
Results
FGF-10 gene expression
IHC detection of FGF-10 protein
Figure 2A depicts a representative RT-PCR gel showing FGF-10 gene expression in the fetal and adult ovaries. The transcripts for FGF-10 were present in all adult and fetal samples tested. Findings for the control b-actin gene were positive in all samples tested (Figure 2B). None of the negative controls processed without RT yielded an amplification product (Figure 2A and B, lane 9).
Table 1 summarizes the IHC findings for the expression of the FGF-10 protein in ovarian samples from fetuses, girls and women. Positive red-brown staining was detected in all the samples tested. In the oocyte, findings were subclassified into nuclear staining and cytoplasmic staining. Positive oocyte staining was described as full (staining in the whole cytoplasm) or partial. An absence of red-brown
Table 1 Fibroblast growth factor 10 protein expression by immunohistochemistry in human ovaries. Source
Level of expression
Fetus Oocyte Cytoplasm Nucleus GC Stroma Girls/women Oocyte Cytoplasm Nucleus GC Stroma
Weak 50% + + (full) 30% + + Weak–medium + + (full) + Weak +
GC = granulosa cells; – = no staining; + = staining; full = full staining.
In the IHC samples from fetuses, 1572 follicles were counted: 1445 (92%) primordial, 126 (8%) primary and one (0.1%) secondary. In the IHC samples from girls and women, 871 follicles were counted: 706 (81%) primordial, 153 (18%) primary and 12 (1%) secondary.
Discussion As far as is known, this is the first study to provide information on the presence of FGF-10 in human preantral follicles from fetuses, girls and women. The IHC results revealed the expression of FGF-10 in oocytes in 50% of the fetal samples (without nuclear staining) and weak GC staining in 30%. In girls and women, positive staining was detected in the oocyte (with nuclear staining) and in the GC in all samples. From RT-PCR, FGF-10 mRNA transcripts were present in all specimens from fetuses and women. Age-related developmental changes can explain the stronger expression and wider distribution of the FGF-10 protein in the samples from girls and women than in samples from fetuses, as well as the identification of oocyte nuclear staining only in the samples from girls and women. Lack of oocyte nuclear staining in samples from fetuses is in line with similar results in previous studies on expression absence of other growth factors and their receptors in human fetal oocyte nuclei (Abir et al., 2010; Ben-Haroush et al., 2005; Pinkas et al., 2008). In general, the present study found a good correspondence between FGF-10 protein and mRNA expression. However, protein staining was weak in the fetal samples, whereas the corresponding mRNA transcripts were easily identified by RT-PCR. This finding might
Fibroblast growth factor 10 in human ovaries
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Figure 1 Immunohistochemistry micrographs of fibroblast growth factor 10 (FGF-10) protein expression. (A) Section of human ovary from a 27-gestational-week-old fetus. Note the primordial follicles, the red-brown staining indicating FGF-10 in oocytes (full cytoplasmic but not nuclear staining) with staining in the GC and staining in a portion of the stroma cells. (B) Section of human ovary from a 13-year-old girl. Note the secondary follicle with the red-brown staining indicating FGF-10 protein in the oocyte (full cytoplasmic and nuclear staining), and in the GC and a portion of the stroma cells. (C) Negative control for the same ovarian section as A: note the primordial follicles with the overall blue staining and the lack of red-brown staining. (D) Negative control for the same ovarian section as B: note the primordial follicles with the overall blue staining and the lack of red-brown staining. Bars = 30 lm.
Figure 2 Representative reverse-transcription PCR gel illustrating fibroblast growth factor 10 (FGF-10) gene expression in fetal and adult ovaries: (A) FGF-10 (308 bp); (B) b-actin control (274 bp). Lanes: 1 = 100 bp DNA ladder; 2 = 21-year-old woman; 3 = 29-year-old woman; 4 = 39-year-old woman; 5 = 21-gestational-week-old fetus; 6 = 22-gestational-week-old fetus; 7 = 25-gestational-week-old fetus; 8 = 27-gestational-week-old fetus; 9 = negative PCR control.
be attributable to a higher RT-PCR sensitivity to low levels of mRNA transcripts relative to the IHC sensitivity to the relevant protein. Be that as it may, the RT-PCR assay was qualitative and not quantitative and, therefore, could not detect changes in mRNA levels. There is very limited information regarding the connection between FGF-10 and mammalian preantral follicles. The present results are in line with a study in cows, wherein
the FGF-10 protein was detected in oocytes of preantral follicles and FGF-10 mRNA transcripts were detected in oocytes at all developmental stages and also in theca cells (Buratini et al., 2007). The differences in the cellular staining pattern between the bovine (Buratini et al., 2007) and the human (the present study) can be attributed to species differences. Accordingly, Chaves et al. (2010) reported that follicular morphology was maintained, with stimulation of
400 follicular growth from the primordial to primary stage, following in-vitro culture of goat preantral follicles with FGF-10. In an earlier work by this study group, mRNA transcripts for the FGF-10 receptor, KGFR, were identified in human ovaries from fetuses, girls and women by in-situ hybridization (Abir et al., 2009). Five of the fetal samples from that study and six of the samples from girls and women were also tested in the present study for the FGF-10 protein. However, follicular KGFR mRNA was found in only 10% of the original samples (Abir et al., 2009), whereas its FGF-10 protein ligand was identified in a larger proportion of samples. This discrepancy may be explained by the higher sensitivity of IHC to protein than of in-situ hybridization for mRNA transcripts in paraffin sections (Pinkas et al., 2008). Additionally, it is noteworthy that although no specific antibody against KGFR is commercially available, the amino acid sequence within the C-terminal cytoplasmic domain is identical in KGFR and FGFR2 (Abir et al., 2009; de Giorgi et al., 2007; Finch and Rubin, 2004; Rubin et al., 1995; Wilkie et al., 2002). Using an antibody targeted against a peptide mapped within this domain, the present study group has identified the FGFR2 protein in oocytes of preantral follicles from human fetuses and girls/women and in GC of girls/women (Ben-Haroush et al., 2005). Therefore, it is likely that the immunostaining procedure for FGFR2 also picked up the KGFR protein (Abir et al., 2009; Ben-Haroush et al., 2005). This is the group’s third report of the identification of FGF members that might be connected with early folliculogenesis in humans, in particular activation of primordial follicles (Abir et al., 2009; Ben-Haroush et al., 2005). The expression of FGF-2 (basic FGF) and FGF-7 and their receptors in human ovaries have been previously identified in fetuses, girls and women (Abir et al., 2009; Ben-Haroush et al., 2005). This group has also shown that FGF-2 promoted 17-b-oestradiol production in cultured human follicles, and contributed marginally to the activation of human primordial follicles (Garor et al., 2008). The present and previous findings (Abir et al., 2009; Ben-Haroush et al., 2005; Garor et al., 2008) shed light on the contribution of the FGF system to the complex interplay between various growth factors in the initial follicular developmental stages in humans, as also reported for other mammals (Buratini et al., 2005). Moreover, this is the third report connecting FGF-10 and preantral follicles in higher mammals (Buratini et al., 2007; Chaves et al., 2010). Be that as it may, definite conclusions regarding the possible role of FGF-10 in growth promotion of human primordial follicles can be drawn only after FGF-10 is included in their culture medium. Additional studies in humans are needed to clarify the role of FGF-10, other growth factors and their synergistic interactions in preantral follicles.
Acknowledgements The authors are greatly indebted to their laboratory technician, Carmela Felz, for preparation of the histological sections. They are grateful to Gloria Ganzach from the Editorial Board, Beilinson Hospital, Rabin Medical Centre for the English editing, to the staff at the gynaecology ward
G Oron et al. for their help in locating suitable patients and to the Ultrasound Unit for identifying fetal gender (all from Beilinson Hospital, Rabin Medical Centre).
References Abir, R., Nitke, S., Ben-Haroush, A., Fisch, B., 2006. In vitro maturation of human primordial ovarian follicles – clinical significance, progress and methods for growth evaluation. Histol. Histopathol. 21, 887–898. Abir, R., Ben-Haroush, A., Felz, C., Okon, E., Raanani, H., Orvieto, R., et al., 2008. Selection of patients before and after anticancer treatment for ovarian cryopreservation. Hum. Reprod. 23, 869–877. Abir, R., Fisch, B., Zhang, X.Y., Felz, C., Kessler-Icekson, G., Krissi, H., Nitke, S., Ao, A., 2009. Keratinocyte growth factor and its receptor in human ovaries from fetuses, girls and women. Mol. Hum. Reprod. 15, 69–75. Abir, R., Ao, A., Zhang, X.Y., Garor, R., Nitke, S., Fisch, B., 2010. Vascular endothelial growth factor A and its two receptors in human preantral follicles from fetuses, girls, and women. Fertil. Steril. 93, 2337–2347. Basilico, C., Moscatelli, D., 1992. The FGF family of growth factors and oncogenes. Adv. Cancer. Res. 59, 115–165. Beenken, A., Eliseenkova, A.V., Ibrahimi, O.A., Olsen, S.K., Mohammadi, M., 2012. Plasticity in interaction of fibroblast growth factor 1 (FGF1) N terminus with FGF receptors underlines promiscuity of FGF1. J. Biol. Chem. 287, 3067–3078. Ben-Haroush, A., Abir, R., Ao, A., Jin, S., Kessler-Icekson, G., Fldberg, D., Fisch, B., 2005. Expression of basic fibroblast growth factor and its receptors in human ovarian follicles from adults and fetuses. Fertil. Steril. 84, 1257–1268. Bottcher, R.T., Niehrs, C., 2005. Fibroblast growth factor signaling during early vertebrate development. Endoc. Rev. 26, 63–77. Buratini, J., Teixeira, A.B., Costa, I.B., Glapinski, V.F., Pinto, M.G.L., Giometti, I.C., Baros, C.M., 2005. Expression of fibroblast growth factor-8 and regulation of cognate receptors, fibroblast growth receptor-3c and-4 in bovine antral follicles. Reproduction 130, 343–350. Buratini, J., Pinto, M.G.L., Castilho, A.C., Amorim, R.L., Giometti, I.C., Portela, V.M., Nicola, E.S., 2007. Expression and function of fibroblast growth factor 10 and its receptor, fibroblast growth factor receptor 2B in bovine follicles. Biol. Reprod. 77, 743–750. Chaves, R.N., Lima-Verde, I.B., Celestino, J.J.H., Duarte, A.B.G., Alves, A.M.C.V., Matos, M.H.T., Campello, C.C., Name, K.P., Bao, S.N., Buratini Jr., J., Figueiredo, J.R., 2010. Fibroblast growth factor-10 maintains the survival and promotes the growth of cultured goat preantral follicles. Domestic Animal Endocrinol. 39, 249–258. De Giorgi, V., Sestini, S., Massi, D., Ghersetich, I., Torello, L., 2007. Keratinocyte growth factor receptors. Dermatol. Clin. 25, 477–485. Dittrich, R., Lotz, L., Keck, G., Hoffmann, I., Mueller, A., Beckmann, M.W., van den Ven, H., Montag, M., 2012. Live birth after ovarian tissue autotransplantation following overnight transportation before cryopreservation. Fertil. Steril. 97, 387–390. Feigin, E., Freud, E., Fisch, B., Orvieto, R., Kravarusic, D., Avrahami, G., Ben-Haroush, A., Abir, R., 2008. Fertility preservation in female adolescents with malignancies. In: Cancer in Female Adolescents. USA Science Publishers Inc., Hauppauge, NY, pp. 38–101. Finch, P.W., Rubin, J.S., 2004. Keratinocyte growth factor/fibroblast growth factor 7, a homeostatic factor with therapeutic potential for epithelial protection and repair. Adv. Cancer Res. 91, 69–136.
Fibroblast growth factor 10 in human ovaries Garor, R., Abir, R., Erman, A., Felz, C., Nitke, S., Fisch, B., 2008. The in vitro effects of basic fibroblast growth factor on human primordial follicles. Fertil. Steril. 91, 1967–1975. Gougeon, A., 1996. Regulations of ovarian follicular development in primates: facts and hypotheses. Endocrinol. Rev. 17, 121–154. Machado, M.F., Portela, M.F., Price, C.A., Costa, I.B., Ripamonte, P., Amorim, R.L., Buratini Jr., J., 2009. Regulation and action of fibroblast growth factor 17 in bovine follicles. J. Endocrinol. 202, 347–353. Ornitz, D.M., Itoh, N., 2001. Fibroblast growth factors. Genome. Biol. 2, 1–12. Pinkas, H., Fisch, B., Rozansky, G., Felz, C., Kessler-Icekson, G., Krissi, H., Nitke, S., Ao, A., Abir, R., 2008. Platelet-derived growth factors (PDGF-A and -B) and their receptors in human fetal and adult ovaries. Mol. Hum. Reprod. 14, 199–206. Ropiquet, F., Giri, D., Kwabi-Addo, B., Schmidt, K., Ittmann, M., 2000. FGF-10 is expressed at low levels in the human prostate. Prostate 44, 334–338. Rubin, J., Bottaro, D., Chedid, M., Miki, T., Ron, D., Cheon, G., Taylor, W.G., Fortney, E., Sakata, H., Finch, P.W., LaRochelle, W.J., 1995. Keratonocyte growth factor. Cell. Biol. Int. 19, 399–407. Silber, S.J., 2012. Ovary cryopreservation and transplantation for fertility preservation. Mol. Hum. Reprod. 18, 59–67.
401 Steinberg, Z., Myers, C., Heim, V.M., Lathrop, C.A., Rubustini, I.T., Stewart, J.S., Larsen, M., Hoffman, M.P., 2004. FGFR2b signaling regulates ex vivo submandibular gland epithelial cell proliferation and branching morphogenesis. Development 132, 1223–1234. Telfer, E.E., McLaughlin, M., Ding, C., Thong, K.J., 2008. A two-step serum-free culture system supports development of human oocytes from primordial follicles in the presence of activin. Hum. Reprod. 23, 1151–1158. Ware, L.B., Matthay, M.A., 2002. Keratinocyte and hepatocyte growth factors in the lung: roles in lung development, inflammation and repair. Am. J. Physiol. 282, 924–940. Wilkie, A.O.M., Patey, S.J., Kan, S., van den Ouweland, A.M.W., Hamel, B.C.J., 2002. FGFs, their receptors, and human limb malformations: clinical and molecular correlations. Am. J. Med. Gen. 112, 266–278. Declaration: The authors report no financial or commercial conflicts of interest. Received 11 March 2012; refereed 12 June 2012; accepted 11 July 2012.