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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2004-0130
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 1 516-521
Copyright © 2005 by The Endocrine Society

Early Human Preantral Follicles Have Relaxin and Relaxin Receptor (LGR7), and Relaxin Promotes Their Development

Kyoko Shirota, Kayoko Tateishi, Takehiko Koji, Yoshitaka Hishikawa, Toru Hachisuga, Masahide Kuroki and Tatsuhiko Kawarabayashi

Departments of Obstetrics and Gynecology (K.S., T.H., T.Ka.) and Biochemistry (K.T., M.K.), Fukuoka University School of Medicine, Fukuoka 814-0180, Japan; and Department of Histology and Cell Biology, Nagasaki University School of Medicine (T.Ko., Y.H.), Nagasaki 852-8523, Japan

Address all correspondence and requests for reprints to: Kayoko Tateishi, Ph.D., Department of Biochemistry, Fukuoka University of Medicine, 7-45-1, Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan. E-mail: tateishi{at}fukuoka-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
The regulatory mechanisms of early follicle development are not clearly understood. Although relaxin is a peptide that controls cell proliferation and differentiation in many tissues, its role in human follicular development is unclear. In this study we cultured slices of human ovarian cortical tissue in the presence and absence of recombinant human relaxin. Ovarian tissue was obtained by biopsy during gynecological laparotomy or laparoscopy (14 women; mean age ± SEM, 29.0 ± 6.1 yr; range, 17–37 yr). A significantly higher proportion of secondary follicles (14.5% vs. 5.0% in the control group; P < 0.01) and a significantly decreased proportion of primordial follicles (30.1% vs. 47.4% in the control group; P < 0.05) were found in tissues cultured with relaxin for 7 d. Immunocytochemical studies with the anti-C-peptide of prorelaxin and antirelaxin antibodies revealed the localization of relaxin in the oocyte and in flat pregranulosa and granulosa cells of primordial, primary, and secondary follicles. The presence of the relaxin receptor LGR7 was observed in flat pregranulosa and granulosa cells of primordial, primary, and secondary follicles by immunocytochemical and in situ hybridization analyses. These results suggest that relaxin plays a role through its receptor during the early stage of follicle development.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
THE MECHANISMS THAT control the initiation of primordial follicle development and regulate early folliculogenesis have not been clarified. Recent studies have focused on growth factors (1, 2) such as insulin (3), IGF-I (4), Kit ligand (KL) (5), basic fibroblast growth factor (bFGF) (6), growth differentiation factor-9 (7), and bone morphogenetic protein-15 (8). Relaxin is a 6-kDa, two-chain peptide hormone belonging to the insulin superfamily and is produced by reproductive and nonreproductive tissues (9). It is secreted from the corpus luteum during the menstrual cycle and pregnancy (10), and previous studies of relaxin have inferred its role in ovulation and formation of the corpus luteum (11). Relaxin has been shown to be confined to thecal cells of antral and preovulatory follicles and luteal cells in the pig (11), monkey (12), and human (13) by immunohistochemical or molecular biological techniques. Several reports suggested that relaxin is one of the hormones involved in the sequence of events that remodel ovarian connective tissue and facilitate ovarian follicle development in the rat (14) and pig (15). A previous study also demonstrated that relaxin increased the proliferation of granulosa cells in cultures of porcine antral follicles (16). These observations suggest that relaxin plays a role even in the early stage of ovarian follicular development and growth in humans. However, the contribution of relaxin to early developing follicles in the human ovary has not been clarified.

Relaxin is structurally similar to insulin and IGFs. Therefore, it seemed logical to assume that its receptor might belong to the insulin family of receptors, which are membrane-associated tyrosine kinase receptors. In contrast, relaxin was known to cause an increase in the concentration of intracellular cAMP in most of its target tissues (17). Furthermore, pharmacological evidence indicated that inhibitors of tyrosine kinase receptors blocked signal transduction by the relaxin receptor, and that relaxin could induce tyrosine phosphorylation and inhibit the activity of a phosphodiesterase that degrades cAMP (18, 19). Recently, the leucine-rich repeat-containing G protein-coupled receptor, LGR7, was identified in the human ovary (20) and was demonstrated to be the receptor capable of mediating the action of relaxin (21). These results clarified that various functions of relaxin in the human ovary are mediated through a type of receptor distinct from receptors of insulin and IGF family ligands.

The purpose of this study was to investigate the effects of relaxin on human ovarian follicles. Therefore, the effect of recombinant human relaxin on the development of follicles at early stages in cultured ovarian cortical tissue was examined. In addition, the localization of LGR7 in primordial follicles and follicles at early stages of development was examined by immunocytochemical and in situ hybridization analyses to determine whether the effect of relaxin is mediated through the receptor.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
This study was approved by the institutional review board of Fukuoka University School of Medicine and was performed after informed consent was obtained from the subjects and parents of minor subjects.

Patient population

Fourteen female patients who had surgery for benign ovarian neoplasm were recruited for the culture study of ovarian cortical tissues. Five patients had endometrioma, and nine patients had mature teratoma. The average age (mean ± SEM) of the patients was 29.0 ± 6.1 yr (range, 17–37 yr), and all patients had normal menstrual cycles. Ovarian cortical tissues were obtained by biopsy during gynecological laparotomy or laparoscopy, and only the histologically normal part was included in this study.

Tissues samples for studies of immunocytochemistry and in situ hybridization were selected from formalin-fixed, paraffin-embedded tissue samples prepared for diagnosis at Fukuoka University Hospital. Normal ovarian tissue samples were selected from the surgical pathology files. All patients enrolled in this study were premenopausal subjects (aged 17–35 yr); they did not show ovarian histopathological abnormalities, nor were they undergoing hormonal treatment.

Tissue culture and analysis of follicular development

Ovarian tissues were collected into HEPES-buffered {alpha}MEM (Invitrogen Life Technologies, Carlsbad, CA) containing 10% serum substitute supplement (SSS) consisting of human serum albumin (50 mg/ml) and human serum globulins (10 mg/ml; Irvine Scientific, Santa Ana, CA). SSS was used instead of serum to avoid the effects of other growth factors. The biopsy specimen from each patient was cut into small pieces (1–2 mm x 1–2 mm; 1 mm thick). Tissue pieces for the noncultured group were fixed without culture and adjoining tissue pieces for the other two groups were cultured in {alpha}MEM supplemented with 10% SSS, transferrin (6 µg/ml; ICN Biomedicals, Inc., Costa Mesa, CA), penicillin (50 IU/ml), streptomycin (50 µg/ml), and FSH (10 IU/ml; Fertinorm P, Serono, Geneva, Switzerland). FSH was added as a survival factor at the early stage (7, 22). The tissues were transferred to 24-well culture dishes (Corning, Inc., Corning, NY) and cultured in 0.5 ml medium at 37 C in a 5% CO2 humidified incubator. To test the effects of relaxin, recombinant human relaxin (20 ng/ml; Immundiagnostik AG, Bensheim, Germany) was added to the culture medium of one of the two parallel cultures of the same biopsy specimen. Those tissues were cultured for 7 d. Half the volume of medium in each well was replaced every other day with fresh medium with or without relaxin.

Ovarian tissue pieces were fixed in Bouin’s solution for 24 h, then dehydrated in 70% ethanol. After paraffin embedding (Sherwood Medical, St. Louis, MO), the pieces were cut into 4-µm sections and stained with hematoxylin and eosin. The number of follicles at each developmental stage was counted in several sections. Follicles were counted only if the oocyte nucleus was present in the section. At least 10 sections were discarded before mounting the next preparation by considering the diameter of follicles to avoid counting the same follicle twice. The developmental stages of follicles have been previously defined (1). We defined primordial follicles as those containing flat follicular cells, primary follicles as those with one or more cuboidal granulosa cells, and secondary follicles as those in which at least part of the follicle had two or more layers of cuboidal granulosa cells.

Production of antibodies to human relaxin C peptide

A cDNA fragment encoding the C peptide of prorelaxin H2 (23) was prepared by RT-PCR from human placenta tissue and ligated to the EcoRI-XhoI site of the pGEX-4T-1 vector. The glutathione-S-transferase fusion protein was obtained by culturing recombinant bacteria and was purified using glutathione-Sepharose 4B (24). The recombinant protein was injected into rabbits to raise antihuman relaxin C peptide antibodies. The IgG fraction was purified using an affinity chromatography kit (MAb Trap G II, Amersham Biosciences, Uppsala, Sweden) and was used for immunocytochemistry.

Immunocytochemistry

Sections (4 µm thick) of formalin-fixed, paraffin-embedded tissue samples were deparaffined, rehydrated, pretreated with 0.3% hydrogen peroxide in methanol for 10 min, and washed for 15 min in 0.05 M Tris-buffered saline (pH 7.4). Antigen retrieval was performed by microwaving at 98 C for 10 min in water. The sections were blocked with a 1:50 dilution of normal goat serum in Tris-buffered saline with 1% BSA. The sections were incubated with antibody against human relaxin C peptide (1:1000) or against H2 human relaxin (6 µg/ml; Immundiagnostik AG) for 3 h at room temperature in a moist chamber. Antigenic sites were immunostained by using polyvalent antibody coupled to horseradish peroxidase (HRP; DakoCytomation, Carpinteria, CA), and the HRP sites were visualized with 3,3'-diaminobenzidine-tetrahydrochloride (DAB) and H2O2. Negative controls included the replacement of specific antibodies with normal rabbit IgG or with the antibodies preincubated with a 100-fold molar excess of antigens. For another control, immunostaining with an antibody against nonrelated peptide, antineurokinin A antibody (25), was performed. The specificity of the anti-C-peptide antibody was confirmed by staining human corpora lutea known to produce relaxin as a positive control and liver, which is not a source of relaxin, as a negative control (26).

For the detection of LGR7, rabbit anti-LGR7 antibody raised against the 23 C-terminal amino acids 735–757 of human LGR7 (1:80; Phoenix Pharmaceuticals, Inc., Belmont, CA) was used. Antigenic sites were immunostained using polyvalent antibody coupled to HRP, and the HRP sites were visualized with DAB, H2O2, Co2+, and Ni2+. A negative control was performed using normal rabbit serum instead of the primary antibody. Positive staining was evaluated based on the density using an image analyzer (DAB system, Carl Zeiss, Inc., Thornwood, NY).

In situ hybridization for LGR7

Human LGR7 mRNA was detected using an oligo-DNA probe complementary to a fragment of human mRNA. The sequence of the antisense probe was 5'-GCCAAGGGAGCACTTGACATCCTGTCCACCCCCATG-3'. Digoxigenin-labeled in situ hybridization was performed according to the method previously described (27). Briefly, sections were deparaffinized and rehydrated using standard procedures and refixed with 4% paraformaldehyde in PBS (120 mM NaCl, 2.7 mM KCl, and 10 mM phosphate buffer, pH 7.4). The slides were treated with 0.3% hydrogen peroxide in methanol-0.2 N HCl, and digestion was performed with 25 µg/ml proteinase K (Sigma-Aldrich Corp., St. Louis, MO). After fixation for 5 min with 4% paraformaldehyde in PBS, sections were immersed in PBS with 2 mg/ml glycine and kept in 40% deionized formamide in 4x SSC (1x SSC = 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0) until hybridization. Hybridization was performed overnight at 40 C with 1 µg/ml digoxigenin-labeled antisense oligo-DNA dissolved in the hybridization buffer containing 10 mM Tris-HCl (pH 7.4), 0.6 M NaCl, 1 mM EDTA, 1x Denhardt’s solution, 250 µg/ml yeast tRNA, 125 µg/ml salmon testis DNA, 10% dextran sulfate, and 40% deionized formamide. After hybridization, the slides were washed three times with 2x SSC for 3 h and with 0.5x SSC for 2 h at 37 C. Sections were reacted with HRP-conjugated goat antidigoxigenin antibody (Roche, Indianapolis, IN), and the HRP sites were visualized with DAB, H2O2, Co2+, and Ni2+. To evaluate the signal specificity, the digoxigenin-labeled sense probe was hybridized with adjacent sections as a negative control. A competitive study was performed by adding a 100-fold excess of unlabeled homologous probe, and a neutralization study was performed by adding a 100-fold excess of unlabeled sense probe to the hybridization buffer together with the labeled antisense probe. Positive cells were evaluated based on the staining density over the sense probe using an image analyzer (DAB system, Carl Zeiss, Inc.).

Statistical analysis

Results are shown as the mean ± SEM and were analyzed by one-way ANOVA with post hoc analysis by Fisher’s least significant difference test. P < 0.05 was considered to be statistically significant.


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Effect of relaxin on follicle development

The average numbers (mean ± SEM) of follicles counted for the noncultured group, the group cultured without relaxin, and the group cultured with relaxin were 31.2 ± 9.8, 40.6 ± 9.6, and 36.2 ± 7.3, respectively (Fig. 1Go). No significant difference was found among those three groups. However, because there was variation among numbers counted in the biopsy sample obtained from each patient, the number of follicles of each classification stage was expressed as the percentage per total follicles counted for each patient in each group. The proportions of primordial, primary, and secondary follicles in noncultured tissues were 75.1%, 21.4%, and 3.5%, respectively (Fig. 2Go). As shown in Fig. 2Go, the proportions of primordial, primary, and secondary follicles in tissues cultured for 7 d without relaxin were 47.4%, 47.6%, and 5.0%, respectively; those in tissues cultured with relaxin were 30.1%, 55.4%, and 14.5%, respectively. A significantly higher proportion of secondary follicles was found in the relaxin-treated group compared with the group without relaxin, and the decrease in the proportion of primordial follicles was significant in the relaxin-treated group compared with the group cultured without relaxin. A significant difference was not found in the proportion of secondary follicles between the noncultured group and the group cultured without relaxin.



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  		FIG. 1. Numbers of follicles counted in noncultured group and groups cultured without or with relaxin (RLX) for studying the effect of relaxin on follicular development.

 


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  		FIG. 2. Proportion of follicles at the primordial ({square}), primary ({cjs2108}), and secondary ({blacksquare}) stages of development in noncultured tissues and tissues after culture without or with relaxin (RLX) for 7 d. The number of follicles at each stage is expressed as the percentage per total follicles counted for each patient in each group (n = 14). The groups are significantly (P < 0.05) different from each other if columns have different superscript letters, as determined by ANOVA. Data are presented as the mean ± SEM.

 
When the specificity of the effect of relaxin was assessed by adding synthetic relaxin B chain in tissue cultures in a preliminary experiment, there was no difference between the control group treated without relaxin and the group treated with relaxin B chain (data not shown).

Immunocytochemical localization of relaxin

Immunostaining with antirelaxin C peptide antibody was observed in human ovarian follicles at different stages. A positive reactivity was found in oocytes and flat pregranulosa cells in primordial follicles (Fig. 3AGo) and in oocytes and granulosa cells in primary (Fig. 3BGo) and secondary (Fig. 3CGo) follicles. Staining of granulosa and thecal cell layers was observed in preantral and antral follicles, as described by others in previous reports (not shown). A similar pattern of immunostaining was observed when human relaxin-2 antibody was used (Fig. 3FGo). However, the staining was relatively weaker than that with anti-C-peptide. For control experiments, reaction with normal rabbit IgG (Fig. 3DGo) at the same concentration, or preabsorption with recombinant relaxin C peptide (Fig. 3EGo) resulted in a lack of significant immunoreactions in adjacent sections. No positive staining in granulosa cells and oocytes was observed when the antibody against neurokinin A, which is not found in follicles, was used instead of anti-C-peptide antibody (Fig. 3GGo). Human liver, which is not a source of relaxin, did not immunostain (Fig. 3HGo), and the positive staining of human corpora lutea known to produce relaxin is shown in Fig. 3IGo.



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  		FIG. 3. Immunocytochemical localization of relaxin in human follicles. Representative results of experimental slides immunostained with antibodies against human relaxin C peptide (A–C) and H2 human relaxin (F) are shown. A, Primordial follicle; B, primary follicle; C–G, serial sections of secondary follicle. Positive staining was found in oocytes and granulosa cells at each stage of follicular development (A–C and F). No positive staining was observed in the follicle reacted with normal rabbit IgG (D), the follicle reacted with antirelaxin C peptide antibody preabsorbed with its antigen (E), or the follicle reacted with anti-neurokinin-A antibody (G). H, Human liver as a negative tissue control; I, human corpus luteum as a positive tissue control. Scale bar, 50 µm.

 
Immunocytochemical localization of LGR7

LGR7 immunoreactivity was observed in flat follicular cells of primordial follicles (Fig. 4AGo) and granulosa cells in primary (Fig. 4BGo) and secondary (Fig. 4CGo) follicles. Omission of the primary antiserum did not result in any significant immunoreaction (Fig. 4DGo).



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  		FIG. 4. Immunocytochemical localization of LGR7 in human follicles. Granulosa cells are immunolabeled in primordial (A), primary (B), and secondary follicles (C). C and D are serial sections. No positive staining was observed in D, which was reacted with normal rabbit IgG as a negative control. Scale bar, A and B, 20 µm; C and D, 40 µm.

 
In situ localization of LGR7 mRNA in follicles

Positive signals for LGR7 mRNA were detected in the flat follicular cells of primordial follicles (Fig. 5AGo) and in granulosa cells in primary and secondary follicles (Fig. 5Go, B and C). In adjacent sections, no staining was found with the sense probe (Fig. 5DGo), and positive signals were completely abolished in a neutralization study using antisense probe in the presence of an excess of homologous unlabeled oligo-DNA (Fig. 5EGo). Oocytes at the stages examined in Fig. 5Go showed no signal for the mRNA of LGR7. In the antral follicle, LGR7 mRNA staining of granulosa and thecal cell layers was observed (Fig. 6Go, A and D).



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  		FIG. 5. In situ hybridization of LGR7 mRNA in human follicles at early stages. Positive signals were evident in flat follicular cells of primordial follicles (A) and granulosa cells in primary (B) and secondary follicles (C). In the section adjacent to C, no signals were present when a sense probe (D) or an antisense probe plus excess homologous DNA (E) was used. The red color was assigned to positive cells by an image analyzer, and the strength of red is shown in each left column. Scale bar, 40 µm.

 


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  		FIG. 6. In situ hybridization of LGR7 mRNA in human antral follicles. Positive signals were evident in granulosa and thecal cells (A); no signals were present in the adjacent section when a sense probe (B) or an antisense probe plus excess homologous DNA was used (C). Granulosa cells and thecal cells are shown as g and t, respectively. Scale bar, A–C, 50 µm (A is a higher magnification of the box inset in D); D, 500 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
The present study showed that treatment with relaxin promoted the progression of early human follicles, and relaxin was present in those follicles. Its receptor, LGR7, was observed in primordial, primary, and secondary follicles. Expression of LGR7 in the ovary of immature rats (20) and human (28) has been detected by Northern blot analyses, but its localization in the human ovary had not been demonstrated previously. Immunocytochemical and in situ hybridization techniques were used to investigate the localization of LGR7 in human ovarian cells in this study. Our results showed the expression of LGR7 in flat pregranulosa cells of primordial follicles and granulosa cells of primary and secondary follicles. To our knowledge, this is the first report demonstrating the localization of relaxin and its receptors in human ovarian follicles at early stages of development. Treatment of ovarian tissues with relaxin resulted in a significant decrease in primordial follicles and a significant increase in secondary follicles. These results suggest that an intrinsic relaxin signaling system expressed in human preantral follicles operates to control the development of follicles at early stages in the human ovary.

Primordial follicles were transited to the primary stage in our culture system even without relaxin. This observation is consistent with findings obtained using bovine (29) and baboon (30) follicles in vitro. Those studies suggested that although follicle growth initiation in vivo is prevented by an intraovarian inhibitor, cultured primordial follicles in the cortical pieces are released from the influence of inhibitors, or stimulatory factors can override the effect of the inhibitors (2, 29, 30). Supplementation of FSH was required as a survival factor at early stages (22) to reduce levels of atresia. In contrast, it has been demonstrated that the presence of FSH did not affect the proportion of follicles that entered the growth phase (22, 29).

A hypothetical model for the initiation of follicle growth consists of two phases (2). Phase 1 involves the proliferation of granulosa cells, followed by their transformation from flattened to cuboidal in shape under the influence of locally produced inhibitory and stimulatory signals. Currently, it has been suggested that at least four growth factors, such as insulin (31), leukemia inhibitory factor (LIF) (32), KL (5), and bFGF (6), stimulate the primordial to primary follicle transition. KL and LIF are produced by pregranulosa cells and act on the oocyte or thecal cells. bFGF is produced in the oocyte and acts on somatic cells. Insulin is an endocrine-type factor, and the localization of insulin receptors in the oocytes suggests that insulin is probably acting upon the oocyte for the differentiation (33). Insulin has been also found to have an additive effect with KL and LIF, but not bFGF (31). In phase 2, the oocyte commences growth and starts to produce factors essential for additional proliferation of granulosa cells, and granulosa cells produce factors promoting oocyte growth and factors protecting follicles from possible inhibitory effects. The oocyte produces growth differentiation factor-9 and bone morphogenetic protein-15, which are essential for the proliferation of granulosa cells, and granulosa cells secrete KL, which promotes oocyte growth. Thus, the oocyte and granulosa cells form an autonomous unit, and their additional growth depends on follicle-produced factors, rather than local intraovarian signals. The existence of relaxin in the oocyte and pregranulosa cells of primordial follicles and of LGR7 in the pregranulosa cells of these cells suggests that relaxin may take part in the proliferation of granulosa cells, followed by gradual transformation of granulosa cells during phase 1. The localization of LGR7 and relaxin in granulosa cells of primary follicles and of relaxin in oocytes of these follicles suggests that relaxin functions in the additional proliferation of granulosa cells during phase 2 through the receptor in a paracrine and autocrine fashion. Contrasting with insulin, relaxin localizes in oocytes and granulosa cells, and its receptor was detected in granulosa cells, but not in oocytes. This suggests that relaxin acts not directly upon the oocyte, but on the granulosa and adjacent stroma cells by autocrine and paracrine mechanisms involving the developmental process of follicles and also may have a role in stimulating other factors secreted from granulosa cells. Furthermore, the presence of LGR7 in thecal and granulosa cells in antral follicles suggests that relaxin may act on the growth of follicles at later stages, although we could not examine the development of follicles in these stages in our culture system. Thus, relaxin may be involved in follicular development at various stages.

Relaxin has a heterodimeric structure consisting of an A and a B chain produced by intracellular excision of a connecting C peptide as posttranslational processing of a precursor polypeptide. Precursors comprised of a B chain, C peptide, and an A chain may be predominant in tissues. Generally, the half-life of active forms of peptide hormones is shorter than that of their prohormone precursors. Therefore, we believed that anti-C-peptide antibody was more suitable for the detection of relaxin localization in ovarian tissue. In the present study, staining with anti-C-peptide antibody clearly showed its localization in oocyte and granulosa cells, suggesting the production of relaxin precursor at those sites. The use of anti-C-peptide antibody proved that the positive staining colocalized with LGR7 in granulosa cells did not depend on the detection of relaxin bound to receptors.

A number of factors have been shown to influence primordial follicles and/or the recruitment of follicles. These factors are considered to influence interactions between developing and primordial follicles. Identification of the actions of each factor and interactions between them will provide a potential sequence of events detailing the initial process of folliculogenesis and the development of follicles. Also, it will afford a therapeutic target to control follicular development in humans. It is unknown how relaxin influences other factors and what the exact role of relaxin is in the underlying molecular mechanisms occurring in oocytes, granulosa cells, or both. However, the significance of relaxin as one of the factors capable of promoting follicular development during early stages is supported by our current findings.


   Acknowledgments
 
We thank Yumiko Hirose for help with the slide preparation. We are grateful to Dr. Hirotsugu Koga at Nagata Clinic and all the doctors at the Department of Obstetrics and Gynecology, Fukuoka University Hospital, for sample collection.


   Footnotes
 
This work was supported in part by Grants-in-Aid for Scientific Research from Japan Society for the Promotion of Science (to K.T., M.K., and T.Ka.) and funds from the Central Research Institute of Fukuoka University (to K.T.).

First Published Online October 13, 2004

Abbreviations: bFGF, Basic fibroblast growth factor; DAB, 3,3'-diaminobenzidine-tetrahydrochloride; HRP, horseradish peroxidase; KL, Kit ligand; LGR, leucine-rich repeat-containing G protein-coupled receptor; LIF, leukemia inhibitory factor; SSS, serum substitute supplement.

Received January 25, 2004.

Accepted September 28, 2004.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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