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Department of Obstetrics and Gynecology, Tottori University School of Medicine, Yonago 683, Japan
Address all correspondence and requests for reprints to: Dr. Masahiro Tanikawa, Department of Obstetrics and Gynecology, Tottori University School of Medicine, Yonago 683, Japan.
Abstract
The c-kit protooncogene receptor and its ligand stem cell factor (SCF) regulate the proliferation and survival of germ cells as well as hematopoietic cells and melanocytes. In adult rodent ovary, c-kit and SCF play important roles in follicular development. However, little information about c-kit in the human ovary is available. In this study, we examined the expressions of c-kit messenger ribonucleic acid (mRNA) and c-kit protein in human oocytes, granulosa cells, and follicular fluid obtained from the women who underwent in vitro fertilization or laparoscopic examination. Expression of c-kit mRNA was detected by RT-PCR in the oocytes and granulosa cells. Western blot analysis showed the presence of soluble c-kit protein in the follicular fluid, and lower levels of c-kit protein were detected in the granulosa cells and the supernatant of granulosa cell cultures. The concentration of soluble c-kit in follicular fluid measured by enzyme-linked immunosorbent assay showed significant correlation with fluid volume and follicular fluid concentrations of estradiol, testosterone, and androstenedione. In summary, we found for the first time the presence of c-kit mRNA and soluble c-kit protein in human oocytes and follicular fluid. The results suggested that in human ovary, c-kit may play an important role in follicular development.
MUTATIONS at either the white spotting (W) or steel (Sl) locus in mice cause defects in germ cell development, melanogenesis, and hematopoiesis. Genetic mapping experiments have revealed that the W locus encodes the c-kit protooncogene, a receptor molecule with tyrosine kinase activity (1), and the Sl locus encodes its corresponding ligand stem cell factor (SCF) (2). These genes regulate signal transduction mechanisms in several types of cells. c-kit and SCF play important roles in the survival and proliferation of the primordial germ cell and in ovarian follicular growth. During embryonic development, c-kit messenger ribonucleic acid (mRNA) is expressed in the primordial germ cell, whereas SCF transcript is expressed along their migratory pathway toward the genital ridge (3). In the mouse ovary, inhibition of c-kit function was found to disturb specific stages of follicular development (4). In the postnatal rodent ovary, the c-kit receptor is detected in the theca cells and oocytes (4, 5, 6), whereas SCF is detected in granulosa cells (6, 7, 8). Although SCF and c-kit may also play essential roles in human ovaries, little information is available.
Recently, soluble forms of several cytokine receptors have been identified and shown to play a role in modulation of cytokine activity (9). However, no information is available about soluble c-kit in human follicular fluid. In the present study, we examined the expressions of c-kit mRNA in human oocytes and granulosa cells, and of c-kit protein in follicular fluid and granulosa cells. To determine the physiological role of c-kit protein, we also investigated the relationship between soluble c-kit and steroid hormone concentrations in follicular fluid.
Subjects and Methods
Subjects
After obtaining informed consent, follicular fluid was collected from five women (mean age ± SD, 33.6 ± 1.1 yr; range, 32–35 yr) who were undergoing oocyte pick up for in vitro fertilization and embryo transfer (IVF-ET) and from nine women (mean age ± SD, 35.4 ± 6.5 yr; range, 22–42 yr) who underwent laparoscopic examination for infertility. Indication for IVF-ET was tubal factor (for four patients) and endometriosis (for one patient). Those undergoing IVF-ET were stimulated with a combination of a GnRH analog and hMG (10). A dose of 5000 IU hCG was administered when the majority of follicles had reached 16 mm in diameter, and the concentration of serum estradiol (E2) per each large follicle exceeded 200 pg/mL. Aspiration of follicular fluid and collection of oocytes were performed 36 h after the administration of hCG. The number of follicle aspirated was 7.4 ± 2.3 (range, 5–11), the number of oocyte retrieved was 6.0 ± 1.8 (range, 3–8), and the E2 level at the time of hCG treatment was 2791 ± 1061 pg/mL (range, 1120–3860 pg/mL). Laparoscopy was performed in the proliferative phase of the cycle.
RT-PCR
Human oocytes and preovulatory granulosa cells were collected from the women who were undergoing IVF-ET. Seven oocytes were used in this study. These oocytes were donated for research purpose by the patient. Surrounding cumulus cells were removed completely by hyaluronidase treatment and mechanical dissection. All oocytes used in this study were metaphase II. Oocytes were stored in the guanidine thiocyanate solution (Nippon Gene Co., Tokyo, Japan) immediately after they have been collected. Granulosa cells were enzymatically dispersed and separated from red blood cells by centrifugation through 45% Percoll gradients (Pharmacia Fine Chemicals, Milton Keynes, UK). Granulosa cells were cultured in DMEM supplemented with 10% FCS, 2 mmol/L L-glutamine, and antibiotics (100 IU/mL penicillin and 100 µg/mL streptomycin) at 37 C in a 95% air-5% CO2 humidified environment. A human erythroleukemia cell (HEL) line was used as a positive control. HEL cells were adapted for growth and maintained in RPMI 1640 medium supplemented with 10% FCS.
Total RNA was extracted from the oocytes, cultured granulosa cells, and HEL cells by the guanidium thiocyanate method (11). RT of RNA into complementary DNA (cDNA) and PCR amplification were performed using a Gene-Amp RNA PCR Core Kit (Perkin-Elmer, Norwalk, CT). Specific primers for c-kit, synthesized as previously described (12), were 5'-AAGGACTTGAGGTTTATTCCT-3' (sense) and 5'-CTGACGTTCATAATTGAAGTC-3' (antisense); these primers amplified a product of 345 bp. Specific primers for ß-actin, synthesized as previously described (13), were 5'-GTGGGGCGCCCCAGGCACCA-3' (sense) and 5'-CTCCTTAATGTCACGCACGATTTC-3' (antisense); these primers amplified a 548-bp product. Amplification was performed for 30 cycles of denaturation (30 s at 94 C), annealing (30 s at 60 C), and synthesis (90 s at 72 C). The specificity of the PCR product was confirmed by Southern blot analysis using the biotinylated oligonucleotide internal probe (5'-CTGTCTGCATTGTTC-3') (14).
Western blotting
For protein extraction, samples were homogenized in lysis buffer (50 mmol/L Tris-HCl, 125 mmol/L NaCl, 0.1% Nonidet P-40, 5 mmol/L ethylenediamine tetraacetic acid, 50 mmol/L NaF, 0.1% phenylmethylsulfonylfluoride, and proteinase inhibitor) and centrifuged at 25,000 x g for 5 min. The total protein concentration in each supernatant was measured, and samples of 30 µg protein were separated by electrophoresis on a 5–15% gradient polyacrylamide gel. The separated proteins were transferred to a polyvinylidene difluoride membrane (Millipore Co., Bedford, MA), which was incubated for 2 h with mouse antihuman c-kit monoclonal antibody (Nichirei Corp., Tokyo, Japan). This commercial c-kit antibody was synthesized with human c-kit cDNA. Specific binding to human c-kit was confirmed by Western blotting using BALB/3T3 cells transfected with human c-kit cDNA (15). The immunoreactive proteins were visualized using peroxidase-conjugated rabbit antimouse polyclonal antibody as a second antibody.
Assay for soluble c-kit, E2, progesterone (P), testosterone (T), and androstenedione (A)
The concentrations of soluble c-kit were determined by means of specific sandwich enzyme-linked immunosorbent assay kit (Nichirei Corp., Tokyo, Japan) as described previously (15). The detection limits of the enzyme-linked immunosorbent assays were 6.25 arbitrary units/mL, and the intraassay variations were less than 8%. The concentrations of E2 and P were measured by enzyme immunosorbent assay kit (Amerlite E2 and Amerlite P, Johnson & Johnson, Clinical Diagnostics, Amersham, Aylesbury, UK). The detection limits of the enzyme immunosorbent assay for E2 and P were 13 pg/mL and 0.1 ng/mL, respectively, and the intraassay variations for E2 and P were less than 13% and less than 11%, respectively. The concentrations of T and A were measured by RIA kit (Diagnostic Products Corp., Los Angeles, CA). The detection limits of this kit for T and A were 4 and 0.04 ng/mL, respectively, and the intraassay variations for T and A were less than 9% and less than 12%, respectively.
Statistical analysis
Correlation analysis was performed with linear regression analysis. P < 0.05 was accepted as statistically significant.
Results
RT-PCR analysis showed the presence of c-kit mRNA in
the oocytes and granulosa cells collected from the women who underwent
IVF-ET (Fig. 1
). The presence of 95-kDa
soluble c-kit protein in follicular fluid was found by
Western blot analysis. We also observed the expression of soluble
c-kit protein in the supernatant of granulosa cell cultures.
However, its expression in the supernatant was low. In consistent with
the findings, cultured granulosa cells expressed a low level of the
145-kDa transmembrane receptor protein (Fig. 2).
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The E2/androgen ratio in follicular fluid is demonstrated
in Fig. 3. E2/androgen ratios
in the fluid indicated that most of follicles in this study were
estrogen dominant. We observed significant correlations between
c-kit and follicular fluid volume and between
c-kit and E2, T, and A concentrations (Figs. 4 and 5).
In contrast, we found no correlation between c-kit and P
concentrations (y = 7.1x + 129.2; r =
0.16; P = 0.31).
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It has been reported that mutations of human c-kit cause congenital white spotting or piebaldism, suggesting that the function of human c-kit is similar to that in mice (16). However, little information about the role of c-kit in human follicular development is available. Horie et al. reported the localization of c-kit protein in the human reproductive organs, and c-kit protein was detected immunohistochemically in the oocyte of secondary follicles (17). The present study demonstrated the expression of c-kit mRNA in human oocytes and granulosa cells. The ligand for c-kit, SCF, mRNA has been detected in the human granulosa cells (7). Thus, c-kit may play an important role in the development of human follicles and oocytes in an autocrine and/or paracrine manner. Although immunohistochemical or in situ hybridization studies are necessary to reveal c-kit protein or mRNA expression during oocyte and follicle maturation, the limited number of human materials is a major problem in such experiments.
Soluble c-kit has been detected in healthy human serum, and the serum c-kit level has been reported to reflect the pathological states of patients with various hematological disorders (15). On the other hand, there was no report about soluble c-kit in human reproductive organs. In this study, we detected soluble c-kit protein in follicular fluid. The result of Western blotting showed lower expression of c-kit protein in the granulosa cells and its supernatants, suggesting that granulosa cells are not the major source of soluble c-kit in follicular fluid.
The current results demonstrate the presence of soluble c-kit protein in human follicular fluid and showed that the concentration of this protein correlates with follicular fluid volume and E2 concentration, both of which increase during follicle and oocyte maturation (18, 19). Strong correlations were found between soluble c-kit and androgen concentrations. It has been reported that an increased E2/androgen ratio was reflective of healthy ovarian follicles, and hence, higher concentrations of androgens indicated atretic changes (20, 21). In our study, most of the follicles were estrogen dominant follicles, as demonstrated by the E2/androgen ratio. A recent study demonstrated that SCF stimulated theca cell growth and A production (22). These results together with our findings suggest that c-kit may have roles in steroid hormone production during follicular development and that thecal cells and oocytes may be sources of soluble c-kit.
We demonstrated for the first time the expression of c-kit mRNA in human oocyte and granulosa cells and the presence of soluble c-kit protein in follicular fluid. Further studies are necessary to elucidate the functional role of interactions between c-kit and its ligand in the regulation of human follicular development.
Received July 16, 1997.
Revised December 22, 1997.
Accepted January 12, 1998.
References
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  K. J. Hutt, E. A. McLaughlin, and M. K. Holland KIT/KIT Ligand in Mammalian Oogenesis and Folliculogenesis: Roles in Rabbit and Murine Ovarian Follicle Activation and Oocyte Growth Biol Reprod, September 1, 2006; 75(3): 421 - 433. [Abstract] [Full Text] [PDF]
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x174 DNA size markers were used (lane
M). B, The specificity of PCR products was confirmed by Southern blot
analysis. Lane 1, HEL cells; lane 2, granulosa cells; lane 3, oocytes.
Results are representative of three experiments.
