Molecular Human Reproduction, Vol. 7, No. 9, 845-852,
September 2001
© 2001 European Society of Human Reproduction and Embryology
Embryology |
Germ cell specific expression of c-kit in the human fetal gonad
MRC Human Reproductive Sciences Unit, Centre for Reproductive Biology, 37 Chalmers Street, Edinburgh EH3 9ET, UK
Abstract
The proto-oncogene receptor, c-kit, and its ligand have been demonstrated to be essential to the processes of germ cell migration, proliferation and survival in the rodent. The aim of the present study was to investigate the expression of c-kit mRNA and protein in human fetal ovary and testis across the gestational period 13–21 weeks. In the ovary, this crucial period of development spans the transition from oogonial replication by mitosis to primordial follicle formation. In the testis, germ cells (gonocytes) are mitotically active. Expression of c-kit mRNA was demonstrated by reverse transcription–polymerase chain reaction (RT–PCR) in both ovary and testis at all gestational ages examined. Testicular germ cell specific expression of c-kit mRNA was confirmed by RT–PCR using specific cell types recovered by laser capture microscopy. The expression of c-kit protein by both male and female germ cells was demonstrated by immunohistochemistry at all gestational ages examined, and was confirmed by immunoblotting. In both, c-kit was localized to the cell membrane except in oocytes within primordial follicles where it was localized to the cytoplasm. These data demonstrate that the expression of c-kit mRNA and protein is germ cell specific in human fetal gonads and are consistent with an important role for the c-kit/kit ligand signalling system in germ cell proliferation and survival in the developing human gonad.
c-kit/gonocyte/human/oocyte/laser capture microscopy
Introduction
In the human the genital ridge destined to develop into either a testis or an ovary appears as a thickening of the intermediate mesoderm at 4 weeks and remains identical in males and females until the seventh week. At 8 weeks the testis and ovary can be distinguished morphologically from each other and the testis can be seen to contain testis cords containing Sertoli cells surrounded by an interstitium which includes Leydig cells (Gilbert, 1997
). Germ cells of vertebrate species do not initially form within the genital ridge but originate in the extraembryonic mesoderm of the yolk sac. Primordial germ cells increase by mitosis during migration and become associated with the cells within the gonadal ridges at ~6 weeks (Byskov, 1986). During fetal life, male germ cells continue to proliferate up to about week 22 of gestation (Hilscher, 1991). Within the fetal ovary, following oogonial replication by mitosis, nests of syncytial germ cells form and are linked by cytoplasmic bridges. The germ cells subsequently enter meiosis only to arrest at diplotene of the first meiotic division (Hilscher, 1991). At this time point, the germ cells become surrounded by somatic cells, thus forming primordial follicles. This process, allowing communication between oocyte and somatic cell, is believed to be crucial for the survival of primordial follicles which may be required to remain in that arrested state for up to 50 years (Gosden, 1995).
Studies in rodents have highlighted the importance of the c-kit proto-oncogene receptor and its ligand, the kit ligand (stem cell factor), in migration of germ cells from the yolk sac to the developing gonad and in their subsequent survival and development (Manova et al., 1990; Godin et al., 1991; Pesce et al., 1993). For example, analysis in mice of the effects of mutations of the White Spotting and Steel loci (encoding c-kit and the kit ligand respectively) has allowed the demonstration of the importance of this ligand–receptor pair in multiple stem cell lineages including melanogenesis and haematopoiesis as well as gametogenesis (Besmer, 1991; Ashman, 1999). c-kit is predominantly expressed by germ cells in the rodent testis although it has also been suggested to be expressed by Leydig cells (Manova et al., 1990), whereas kit ligand is expressed by a wider range of cell types. The presence of a functional c-kit receptor has been implicated in spermatogonial proliferation, survival and adhesion to Sertoli cells (Loveland and Schlatt, 1997). Vincent et al. have demonstrated expression of c-kit by pachytene spermatocytes and proposed that the kit/kit ligand interaction is essential for meiosis (Vincent et al., 1998).
mRNA encoding c-kit and kit ligand have been detected in fetal mouse ovaries between embryonic days 8 and 14.5, consistent with a role in germ cell migration and proliferation (Driancourt et al., 2000). In their review, Driancourt and colleagues used ovaries from mice in which one copy of the kit gene was replaced by a lac-z reporter construct (Bernex et al., 1996) to demonstrate that c-kit mRNA is not present in oogonia in fetal ovaries on day 15.5, but is transcribed at high levels in oocytes in primordial and growing follicles. These results are in agreement with the other findings (Manova et al., 1990) using in-situ hybridization studies. Functional effects of kit ligand/c-kit in the ovary may persist into adult life, for example in the regulation of persistence of meiotic arrest (Horie et al., 1991; Ismail et al., 1997) and activation of primordial follicle growth (Yoshida et al., 1997; Parrott and Skinner, 1999).
Studies identifying the sites of expression of c-kit in the human fetus have been very limited compared with those in rodents and there are inconsistencies between the results so far reported. Horie and co-workers used specific immunohistochemistry to detect c-kit protein on frozen sections from a number of human tissues, and in their paper immunopositive staining of single sections from a human fetal testis (18 weeks) and human fetal ovary (20 weeks) was shown (Horie et al., 1993). However, other investigators have suggested that c-kit is not detectable in the fetal testis after 15 weeks gestation (Rajpert de-Meyts et al., 1996). It is notable that in one study (Rajpert de-Meyts et al., 1996), c-kit remained detectable in intersex testes until later in gestation, and other studies from the same group have demonstrated that c-kit is a marker of carcinoma in situ (CIS), a pre-malignant lesion thought to be associated with persistence of fetal-type germ cells in the adult testis (Rajpert de-Meyts and Skakkebaek, 1994). Consistent with the suggestion that signalling via c-kit is important in normal male germ cell development and function, alterations in c-kit/kit ligand expression have also been demonstrated in some patients with defective spermatogenesis (Mauduit et al., 1999), with reduced expression associated with increased germ cell apoptosis.
As the second trimester is the major time for the regulation of germ cell numbers in the female (Baker, 1963) and is a period of continuing testicular development (Wartenberg, 1989), we have examined the expression and localization of c-kit in the human gonad between 13 and 20 weeks of development. Our studies have demonstrated that c-kit mRNA and protein are expressed specifically in germ cells of both sexes during this critical period.
Materials and methods
Tissues
Human fetal gonads were obtained following medical termination of pregnancy. Women gave consent according to national guidelines (Polkinghorne, 1989) and the study was approved by the Lothian Paediatrics/Reproductive Medicine Research Ethics Sub-Committee. Termination of pregnancy was induced by treatment with mifepristone (200 mg orally) followed by prostaglandin E1 analogue (Gemeprost; Beacon Pharmaceuticals, Tunbridge Wells, UK) 1 mg 3 hourly per vaginam. None of the terminations were for reasons of fetal abnormality, and all fetuses appeared morphologically normal. Gestational age was determined by ultrasound examination prior to termination and confirmed by subsequent direct measurement of foot length. A total of 20 specimens was used for this study, divided equally between male and female.
Ovaries and testes were dissected free, and either fixed for immunohistochemical analysis or snap-frozen and stored at –70°C. Fixation was carried out in Bouin's fluid for 5 h, followed by transfer to 70% ethanol prior to processing into paraffin using standard methods.
Isolation of RNA and synthesis of cDNA from whole tissues
Total RNA was extracted from snap-frozen samples of fetal ovary (13–21 weeks, n = 10) and testis (14–19 weeks, n = 6) using the RNeasy mini kit (Qiagen, Crawley, UK). RNA was treated with DNase (Gibco, Paisley, UK) and reverse transcription performed using a first strand cDNA synthesis kit (Roche Diagnostics, Lewes, UK). Briefly, 1 µg total RNA was incubated with oligo (dT)18 primer for 10 min at 65°C and then placed on ice. A reaction mix comprising buffer, 1 mmol/l each deoxynucleotide triphosphate (dNTP), ribonuclease inhibitor and 50 IU reverse transcriptase, was added to each tube in a total volume of 50 µl and the tubes were then incubated at 40°C for 2 h.
Isolation of RNA by laser capture microscopy
Sections (5 µm) were cut from paraffin wax-embedded 19 week human fetal testis samples and mounted on plain, uncoated, glass slides. Sections were dewaxed in xylene, rehydrated then subjected to immunostaining. To visualize the cells within the seminiferous cords, an anti-Müllerian hormone (AMH) polyclonal antibody was used as detailed below except that the protocol was modified for short, typically 10 min, incubation times at each step to reduce the chance of RNA degradation, and RNAse inhibitor (200 IU/ml, Promega) was included in all the immunohistochemical reagents. After colour development with diaminobenzidine (DAB), the sections were dehydrated through graded alcohols and finally xylene. Sections were stored in a vacuum desiccator for at least 30 min prior to capture. Care was taken throughout to avoid RNase contamination of sections and all aqueous solutions were prepared with DEPC-treated water.
Individual cell fragments were recovered from the stained sections by microdissection using the PixCell II LCM system (Arcturus Engineering Inc., Mountain View, CA, USA) according to the manufacturer's instructions. Briefly, each section was overlaid with a thermoplastic membrane mounted on optically transparent caps and cell fragments were captured by focal melting of the membrane due to laser activation (Figure 1). The parameters of the laser shot used in this study were: spot size 7.5 µm in diameter, power 45 mW and duration time 0.5 ms. The same parameters and number of laser shots (~600) were used for each cell type to normalize the amount of cellular material isolated.
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Total RNA was extracted from micro-dissected samples with the Micro RNA Isolation Kit (Stratagene, La Jolla, CA, USA). After incubation with 200 µl of denaturing buffer and 1.6 µl of ß-mercaptoethanol at room temperature for 10 min, the sample was extracted with 20 µl of 2 mol/l sodium acetate, 220 µl phenol and 60 µl chloroform:isoamyl alcohol (24:1). The aqueous phase was mixed with 1 µl of 10 mg/ml carrier glycogen and then precipitated with 200 µl of isopropanol. After a 70% ethanol wash followed by drying in air, the pellet was resuspended in 10 µl of RNase free H2O. The extracted RNA was reverse transcribed using 10 pmol random hexamer primers and 200 IU of Superscript II (Gibco BRL) reverse transcriptase according to the manufacturers instructions. An aliquot of cDNA was then amplified using a modified degenerate oligonucleotide primed polymerase chain reaction (DOP–PCR) protocol (Kasai et al., 2000
) using the primer UN1, 5'-CCGACTCGAGNNNNNNATGTGG-3' in a total volume of 25 µl. 5 µl of amplified cDNA was then used in subsequent PCR reactions using primer sets for specific sequences.
Amplification of specific cDNA by PCR
c-kit
PCR was performed by incubating either 1 µl (whole tissue extracts) or 5 µl (samples from LCM) cDNA with Taq DNA polymerase (AGS Gold; Hybaid, Ashford, UK) in buffer containing 0.2 mmol/l of each dNTP and forward and reverse oligonucleotide primers. Two control tubes were run in parallel, one in which water replaced the RNA and a second omitting reverse transcriptase to ensure that there was no genomic DNA contamination. PCR amplification conditions consisted of an initial denaturation step at 95°C for 2 min, followed by 35 cycles of denaturation at 95°C for 30 s, annealing at 50°C for 30 s and extension at 72°C for 45 s; a final extension period at 72°C for 5 min completed the amplification. Three sets of primers specific for human c-kit (Vandenbark et al., 1992) were used, one for cDNA from whole tissue extracts (set 1), the other two following laser capture of samples [sets 2 and 3 (nested)]. All three pairs of primers were designed to span an intron to ensure that genomic DNA was not amplified. Set 1, 5'-AAGGACTTGAGGTTTATTCCT-3' (sense) and 5'-CTGACGTTCATAATTGAAGTC-3' (antisense), amplified a product of 345 bp; set 2, 5'-GTGGTTAAAGGAAACGCTCG-3' and 5'-CATACATTTCAGCAGGTGCG-3', amplified a product of 232 bp; set 3, 5'-AAGTGGATGGCACCTGAAAG-3' and 5'-GAACTTAGAATCGACCGGCA-3', amplified a product of 138 bp from within the product of primer set 2. Primers for the constitutively expressed gene GAPDH were used to confirm the integrity of the RNA and efficacy of the PCR reaction. The identity of all PCR products were confirmed by direct sequencing using an Applied Biosystems 373A automated sequencer.
Anti-Müllerian hormone (AMH)
The following primers were used to identify samples containing Sertoli cell mRNA recovered by LCM (all primers were based on the sequence of human AMH; accession no. NP000470): set 1, 5'-TGCAACACCGGTGACAGGCAG-3' and 5'-GCAGCCCAGCCCTCGTCACAG-3', amplified cDNA 238 bp; set 2 (nested), 5'-GCTGCCTTGCCCTCTCTAC-3' and 5'-GAACCTCAGCGAGGGTGTT3', amplify a product of 117 bp from within the product of primer set 1.
Immunohistochemistry
Sections (5 µm) were mounted on TESPA (Sigma, Poole, Dorset)-coated slides, dewaxed and rehydrated. Endogenous peroxidase activity was inhibited by incubation in 3% H2O2 in methanol for 30 min. After a wash in water, slides were transferred into Tris-buffered saline (TBS; 0.05 mol/l Tris, 0.85% NaCl, pH 7.6) for 5 min and blocked for 30 min in normal rabbit serum (NRS; Diagnostics Scotland, Carluke, UK) diluted 1:4 in TBS containing 5% bovine serum albumin (NRS/TBS/BSA). Sections were then blocked with avidin (0.01 mol/l; 15 min) and biotin (0.001 mol/l; 15 min; both from Vector, Peterborough, UK) with washes in TBS in between. The primary antibody (anti-c-kit goat polyclonal; cat. No. M14 Santa Cruz) was applied at a dilution of 1 in 300 in NRS/TBS/BSA at 4°C overnight. Sections were washed and incubated for 30 min with biotinylated rabbit anti-goat antibody (Dako, Cambridge) diluted 1:500 in NRS/TBS/BSA. Following washes in TBS, sections were incubated with avidin–biotin–horseradish peroxidase linked complex (Dako) according to the manufacturer's instructions. Bound antibody was visualized using 3,3'-diaminobenzidine tetrahydrochloride (Dako). A second anti-c-kit primary antibody (rabbit polyclonal, dilution 1:30; Dako) was also used in some experiments. Testis sections were stained with AMH primary antibody (rabbit polyclonal, gift of Dr R.Rey, Buenos Aires, Argentina) used at a dilution of 1:500 following antigen retrieval using citrate buffer (0.01 mol/l, pH 6.0, pressure cooked for 2.5 min). Swine anti-rabbit second antibody (Dako) was used in both cases. Primary antibodies were omitted as negative controls.
Sections were counterstained with haematoxylin, dehydrated, mounted and visualized by light microscopy. Images were captured using an Olympus Provis microscope (Olympus Optical Co., London) equipped with a Kodak DCS330 camera (Eastman Kodak), stored on a Macintosh PowerPC computer and assembled using Photoshop 5 (Adobe, Mountain View, CA, USA). A total of six ovaries and six testes were examined using immunohistochemistry.
Immunoblotting
Fetal ovaries (n = 2) and testes (n = 4) were homogenized in denaturing buffer [58 mmol/l Tris pH 6.8, 1% sodium dodecyl sulphate (SDS), 1% glycerol; all from Sigma]. Samples (10 µg protein) were diluted with an equal volume of reducing loading buffer (187 mmol/l Tris pH 6.8, 2% SDS, 2% ß-mercaptoethanol, 1% sucrose, 0.01% bromophenol blue) and boiled for 5 min. Proteins were separated by SDS–polyacrylamide gel electrophoresis on a 7.5% acrylamide gel in parallel with prestained protein molecular weight markers (Biorad, CA, USA) and blotted onto PDF membranes (Amersham Pharmacia, Buckinghamshire, UK) overnight using wet blot apparatus (Biorad). Thereafter, membranes were blocked for 2 h at room temperature in 0.02 mol/l TBS (pH 7.6) containing 3% w/v BSA (Sigma). Membranes were washed in TBS with 0.1% Tween-20 (TBST) and then incubated for 2 h with the primary antibody (anti-c-kit goat polyclonal, 1:750, Santa Cruz) in TBST with 1% BSA. Primary antibody was omitted as a negative control. Bound antibody was detected using a rabbit anti-goat HRP linked secondary antibody (1:6000, Dako) and the enhanced chemiluminescence visualization system (Amersham Pharmacia Biotech) according to the manufacturer's instructions.
Results
Expression of c-kit mRNA
By RT–PCR, a single cDNA (345 bp) was amplified from RNA extracted from both fetal ovaries and testes at all gestational ages examined (13–21 weeks) (Figure 2A). Although the PCR was not quantitative, the amount of mRNA detected appeared higher in samples from testis.
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Analysis of cell populations recovered from fixed sections of a week 19 testis by laser capture microscopy showed that at this age expression of c-kit mRNA was confined to the germ cell population (gonocytes) and was not expressed in either Sertoli cells or within the interstitium (Figure 2B–D
).
Immunohistochemistry
c-kit protein was detected by immunohistochemistry in all specimens examined, across the gestational range 14–21 weeks (ovary) and 13–19 weeks (testis) (Figure 3, representative sections from a total of six ovaries and six testes). Both anti-c-kit antibodies tested gave similar staining patterns. In the ovary, positive staining was seen in oogonia and oocytes (Figure 3A–C), with the great majority of germ cells being stained. The surface epithelium, the ovarian stroma and the pregranulosa cells of primordial follicles were all immunonegative. This was particularly apparent in the 21 week gestation specimen in which there were a large number of primordial follicles which were not present at earlier gestations (Figure 3C). It was notable that although the c-kit protein appeared to be concentrated at the cell membrane of the oogonia at earlier gestational ages (Figure 3A and B), at 21 weeks the protein was clearly spread throughout the cytoplasm of the oocytes (Figure 3C).
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Within the testis, c-kit protein was localized to gonocytes within the testicular cords (Figure 3D–F
) consistent with the cell specific expression of c-kit mRNA. Although most gonocytes were immunopositive at all ages examined (13–19 weeks), some immunonegative cells were also present (e.g. 17 weeks, Figure 3F). As with the ovary, immunohistochemical staining was concentrated at the germ cell membrane (Figure 3F). c-kit immunoexpression was not detected in the peritubular cells, interstitial cells (Figure 3F) or the surface epithelium (not shown). Sertoli cells were identified by immunostaining for AMH (Figure 3G). No staining was seen on sections of ovary (Figure 3H) or testis (Figure 3I) in which the primary antibody was not included.
Immunoblotting
The presence of c-kit protein in both fetal ovary and testis was confirmed by immunoblotting. A prominent 145 kDa band corresponding to the size of the transmembrane receptor protein was detected in samples from gonadal tissues of both sexes (Figure 4). There was no immunoreactivity in the absence of the primary antibody. The experiment was repeated three times with similar results.
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Discussion
The results presented demonstrate unequivocally that the expression of c-kit mRNA and protein occurs in the germ cells of both ovary and testis in the human fetus during the second trimester.
In the rodent ovary, expression of the c-kit gene has been detected very early in development and is high in proliferating germ cells (Manova and Bachvarova, 1991); however, expression stops when oogonia enter meiosis (Manova et al., 1990). In the human ovary, entry into meiosis occurs over a wide timescale, being detectable as early as 11 weeks gestation (Gondos et al., 1986) although it is believed to be maximal at ~20 weeks (Baker, 1963). Thus over the range of gestations examined in the present study, many oogonia will continue to proliferate by mitosis while an increasing proportion enter meiosis. The absence of a closely defined time at which mitosis ceases and meiosis is initiated throughout the ovary is likely to account for the detection of c-kit mRNA in whole tissue samples at all gestations examined up to 21 weeks, at which time many primordial follicles were present. The detection of c-kit protein in all specimens examined up to and including 21 weeks gestation is consistent with a previous report in sheep where c-kit protein, but not mRNA, was detected in oocytes undergoing meiosis (Tisdall et al., 1999). It is possible that the apparent change in intracellular localization of c-kit protein from the membrane to the cytoplasm seen in human primordial follicles may be related to this underlying change in gene expression. c-kit protein has been previously localized to the oocytes of a 20 week gestation fetus (Horie et al., 1993), but in that study, which was undertaken on frozen sections, it was unclear whether c-kit was localized to the cytoplasm or cellular membrane.
The range of gestational ages examined in this study covers the period of definitive histogenesis of the ovary, when the finite population of oocytes which will survive within primordial follicles and thus determine reproductive potential is regulated. c-kit and its ligand, stem cell factor or kit ligand, may have an important role in these processes. Mutations in the genes for these factors result in loss of primordial germ cells (Besmer et al., 1993), and effects of kit ligand/c-kit signalling on germ cell survival and protection from apoptosis have been demonstrated in vivo and in vitro (Godin et al., 1991; Pesce et al., 1993; Yee et al., 1994). It has been suggested that the pro-survival effects of c-kit in the ovary may be mediated by increased expression of the anti-apoptotic factor, Bcl-2 (Tilly, 1996). Such studies have also suggested a role for this pathway in regulation of the onset of primordial follicle formation and growth of primary follicles (Yoshida et al., 1997). Kit ligand/c-kit may also have a role in later folliculogenesis: both promotion of follicle development and maintenance of arrest of meiosis have been suggested (Horie et al., 1991; Ismail et al., 1997; Parrott and Skinner, 1999). Kit ligand appears to have effects on surrounding stromal cells in addition to the oocyte (Parrott and Skinner, 2000). c-kit is also present in the adult human ovary, in both oocytes and granulosa cells (Tanikawa et al., 1998) and has been suggested to have an autocrine role in the ovarian surface epithelium (Parrott et al., 2000). c-kit expression appeared to be confined to germ cells within the ovary in the present study: no consistent staining of stromal cells was observed.
The technique of laser capture microscopy (LCM) was developed at the National Institutes of Health in the USA to allow sampling of individual, or groups of cells from complex tissues in such a way that mRNA and/or proteins could be extracted from them and analysed (Emmert-Buck et al., 1996). The method has recently been applied to the recovery of seminiferous tubules from frozen sections of mouse testis (Suárez-Quian et al., 2000). In the present study, the LCM microscope was used with the laser set to the smallest size available (7.5 µm) to allow for sampling of single gonocytes. Recently the size of male germ cells in the human fetal testis (7–10 weeks) has been reported as being 9 µm in diameter (Bendsen et al., 2001). To enable us to locate individual gonocytes, fixed tissue sections were used and these were stained using a modified immunohistochemical technique. The use of fixed sections meant that only short fragments of cDNA could be identified by RT–PCR (Goldsworthy et al., 1999) and we employed a nested PCR strategy to increase signal intensity and specificity. We believe that this is the first time LCM methodology has been used to sample individual cell types from the human fetal testis and it has allowed us to show that c-kit mRNA is expressed in fetal gonocytes at 19 weeks gestation.
In a previous study on human fetal testes, c-kit protein was not detected in the testis beyond 15 weeks gestation using immunohistochemistry (Rajpert de-Meyts et al., 1996). In another study, presence of c-kit in the human fetal testis was reported in cells described as spermatogonia in a fetus of 18 weeks gestation (Horie et al., 1991). The results we have obtained using specific immunohistochemistry and Western analysis all demonstrate that c-kit protein is expressed in the fetal germ cells up to and including 19 weeks of gestation. Gonocytes are believed to be the cell of origin of gonadoblastomas (Jorgensen et al., 1997), and it has been suggested that prolonged expression of c-kit in germ cells in individuals with intersex conditions may be a component of abnormal germ cell development in such individuals who are at increased risk of testicular neoplasia (Rajpert de-Meyts et al., 1996). Following laser capture of cells from a 19 week fetus, we failed to detect expression of c-kit mRNA in interstitial cells. Although this result is based on a single stage of development, it was in agreement with a lack of immunostaining in the interstitium at all ages examined and would not therefore be consistent with findings in the mouse (Manova et al., 1990). Studies of mice in which mutations in c-kit or its ligand have been well documented and result in a failure in migration of germ cells into the genital ridge (Besmer et al., 1993); however, the use of a blocking antibody against c-kit has led to the suggestion that c-kit is important in proliferation of differentiated spermatogonia (Yoshinga et al., 1991) and protection from apoptosis (Packer et al., 1995). A role in spermatogonial differentiation rather than proliferation has also been suggested on the basis of experiments involving transplantation of germ cells into testes of Steel mice (Ohata et al., 2000) and other studies have shown a role for the kit-kit ligand in meiosis (Vincent et al., 1998). Taken together, these data reinforce the importance of c-kit and its ligand in multiple cell lineages both during development and in adulthood.
In conclusion, this study demonstrates conclusively that c-kit mRNA and protein are expressed in oogonia during the transition from rapid proliferation by mitosis to the formation of primordial follicles, and in gonocytes of the developing testis during the second trimester. c-kit has been demonstrated to be crucial for germ cell migration, survival and proliferation in the mouse: the present results suggest that c-kit is likely to be of similar importance in the human.
Notes
1 To whom correspondence should be addressed. E-mail: r.a.anderson{at}hrsu.mrc.ac.uk 
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Submitted on March 22, 2001; accepted on June 28, 2001.
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