Presence of NGF and its receptors in ovaries from human fetuses and adults
1Infertility and IVF Unit, Department of Obstetrics and Gynecology, Rabin Medical Center, Beilinson Campus, Petah Tikva 49100 and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv and 2Department of Obstetrics and Gynecology, Rabin Medical Center, Beilinson Campus, Petah Tikva and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel, 3Department of Obstetrics and Gynecology, Royal Victoria Hospital, McGill University, Montreal, Quebec H3A 1A1, Canada, 4Department of Pathology, Rabin Medical Center, Beilinson Campus, Petah Tikva 49100, 5The Felsenstein Medical Research Center, Beilinson Campus, Petah Tikva 49100 and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel and 6Department of Human Genetics, McGill University, Montreal, Quebec H3A 1A1, Canada
7 To whom correspondence should be addressed at: IVF Research Laboratory, Infertility and IVF Unit, Department of Obstetrics and Gynecology, Rabin Medical Center, Beilinson Campus, Petah Tikva 49100, Israel. E-mail: ronita{at}clalit.org.il
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The ability to mature human primordial follicles in vitro would assist fertility restoration. However, the signals initiating growth of primordial follicles are unknown. Growth factors such as nerve growth factor (NGF) may play a role in this process. To investigate the expression of NGF and its receptors, p75 and TrkA, in early developing follicles (mostly primordial, primary and secondary follicles), ten ovarian samples from adolescents/adults aged 13–39 and 33 ovaries from human fetuses aged 19–33 gestational weeks (GW) were obtained and immediately fixed or frozen. The fixed samples were prepared for a study of immunocytochemical staining of NGF and its two receptors. Total RNA was extracted from the frozen ovarian samples, and the expression of NGF, TrkA and p75 was investigated by RT–PCR. Products were resolved by 1% agarose gel electrophoresis and image analysis. Immunocytochemical staining revealed the expression of NGF in granulosa cells (GC) and oocytes; TrkA was mainly in oocytes and in GC in minority of the samples; and p75 was in some of the stroma cells from fetuses aged less than 22 GW. Transcripts of NGF and TrkA were identified by RT–PCR in all samples, while those for p75 were detected only in ovarian samples from fetuses aged less than 22 GW. To elucidate if NGF is indeed involved in growth initiation of human primordial follicles, it should be added to their culture medium. The immunocytochemical detection of p75 in some of the stroma cells and transcripts in ovarian samples of fetuses less than 22 GW may suggest its role in follicular assembly.
Key words: immunocytochemistry/NGF/primordial follicles/receptors/RT–PCR
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Introduction |
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The germ cells in the fetal ovary before follicular assembly are present in the form of small oogonia (Gosden, 1995
). Human follicular formation begins during gestational weeks (GW) 20–22 when a single flat layer of cells surrounds the oocytes to form primordial follicles. However, in rats and mice, follicular formation is initiated during the first post-natal days (Ojeda et al., 2000). Most of the human ovarian follicles in fetuses as well as adults remain unilaminar (30–50 µm), mostly primordial follicles or primary follicles with cuboidal granulosa cells (GC) (Gougeon, 1996). The signals that trigger their growth into multilayered secondary follicles, with a theca cell layer that differentiates from the surrounding stroma cells, are unknown.
Understanding the signals responsible for the initiation of folliculogenesis is an important step towards developing a successful in vitro maturation system for primordial follicles, a putative assisted reproduction technology that would restore fertility in cancer patients without the risks of reseeding the disease (Abir et al., 1998). It is possible that various growth factors such as nerve growth factor (NGF) might be involved in this process (Van den Hurk et al., 2000). From the secondary stages onwards, FSH induces follicular growth, and the FSH receptor (FSH-R) can be detected already in GC of primary follicles (Gougeon, 1996; Romero et al., 2002).
NGF is a member of the neurotrophin (NT) family (Chao and Hempstead, 1995; Anderson et al., 2002), which is related to the transforming growth factor beta superfamily (Chao and Hempstead, 1995; Dissen et al., 2001; Anderson et al., 2002). NTs are active in the central and peripheral nervous systems, but are also found in non-neural cells, for example, in the ovary (Dissen et al., 2001; Anderson et al., 2002). Their receptor-signalling system is composed of two unrelated transmembrane receptor proteins lacking any sequence similarity: the low-affinity universal NT receptor p75 and the specific high affinity Trk receptor kinases (TrkA, TrkB and TrkC) (Chao and Hempstead, 1995). When p75 and a Trk receptor are co-expressed, p75 increases the sensitivity of the Trk receptor and its signalling efficiency (Ibanez, 1995; Fridman and Greene, 1999; Spears et al., 2003). NGF's receptors consist of both p75 and TrkA (Chao and Hempstead, 1995).
Studies have shown that the NGF and/or its receptors are expressed in oogonia and oocytes from mice (Dissen et al., 2001; Spears et al., 2003), rats (Dissen et al., 2001), rhesus monkeys (Dees et al., 1995), human fetuses (Anesetti et al., 2001; Anderson et al., 2002) and babies (Anesetti et al., 2001). NGF was also found to initiate growth of primordial ovarian follicles from rats (Dissen et al., 1991, 2001; Romero et al., 2002). The aim of the present study was to investigate the expression of NGF and its receptors, p75 and TrkA, in human ovaries from adults and second-to-third trimester fetuses at the RNA as well as the protein level.
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Human adult and fetal ovaries
Approval for the study was obtained from the ethics committee of our institute, and informed consent was obtained from every woman or guardian.
Ovarian samples were obtained from 33 aborted human fetuses aged 19–33 GW (Table I). All but six of the pregnancy terminations were performed because of fetal anatomical malformations or chromosomal abnormalities (Table I) and were induced by prostaglandins. Normal fetuses originated from legal terminations conducted because of psychiatric problems of the mothers. As our department's pregnancy termination policy mandates feticide for all fetuses over 21 GW, only fetal specimens shown to be non-apoptotic previously (Abir et al., 2002) were used in the present study. The use of a deoxynucleotidyl transferase assay for selection of viable fetal ovarian samples has been discussed in detail elsewhere (Abir et al., 2004). Moreover, seven of the 15 samples from fetuses aged 
22 GW were used for culture; the follicles survived for 4 weeks and secreted 17-ß estradiol, further indicating the viability of the specimens (Biron-Shental et al., 2004).
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In addition, small ovarian cortical biopsies were donated by ten premenopausal women and adolescents aged 13–39 years undergoing various gynaecological laparoscopic operations (Table II). Sixty percent of the patients had various forms of cancer. They underwent the operations before chemotherapy for ovarian retrieval for cryopreservation and donated a portion of their tissue for research (Abir et al., 1998
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All visible blood vessels were removed from the specimens. The ovarian samples were cut into uniform size as possible (to approximately 2x2 mm2) and fixed immediately in neutral buffered formalin. A portion of every sample was frozen for subsequent RNA extraction.
Cryopreservation of ovarian tissue
Five to seven tissue slices measuring 1–2 mm were placed in cryogenic vials (Nalge Nunc International, Delta, Roskilde, Denmark) filled with a 1.5 M dimethylsulfoxide (DMSO) (Sigma, St Louis, MO, USA) and 0.1 M sucrose solution (Sigma) (Newton et al., 1998). Prior to freezing, the samples were kept on ice for half an hour. All samples were frozen slowly in a programmable freezer (Kryo 10; series 10/20, Planer Biomed, Sunbury on Thames, UK) and immediately placed in liquid nitrogen. The slices were cryopreserved and stored for 3–24 months until RNA extraction.
Immunocytochemistry for NGF, p75 and TrkA
The fixed specimens were dehydrated in a graded series of ethanol followed by paraffin embedding and sectioning (5 µm). Unstained sections were placed on OptiPlus positive charged microscope slides (BioGenex Laboratories, San Ramon, CA, USA) for immunocytochemistry. The sections were deparaffinized and rehydrated. To enhance antigen retrieval, they were first microwaved for 15 min under full power (800 watts) and then simmered for further 15 min with citrate buffer at pH 6 (diluted in distilled water from a 10x citrate buffer solution at pH 6) (DAKO Corporation, Carpintera, CA, USA). The antigen retrieval procedure was terminated by cooling and rinsing the sections in distilled water and phosphate-buffered saline (PBS) at pH 7.4 (Biological Industries, Beit Ha'Emek, Israel). All sections for the NGF and p75 studies were then quenched in 3% H2O2 (Vitamed, Binyamina, Israel) in the dark to block endogenous peroxidase activity, and rinsed. Sections for the TrkA study were quenched with a peroxidase block containing 0.2% sodium azide (from a DAKO EnVision+System, HRP-AEC anti-rabbit kit, DAKO).
All sections were then incubated with the primary antibodies: rabbit polyclonal antibodies for NGF (antibody concentration 1/150–1/175) and TrkA (antibody concentration 1/60) and a mouse monoclonal antibody for p75 (antibody concentration 1/40) (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Negative control solutions for NGF and TrkA were prepared by absorbing equal volumes of the diluted primary antibody with a 10-fold concentration of the corresponding blocking peptides (Santa Cruz Biotechnology) (diluted 1/15 for NGF and 1/6 for TrkA), followed by incubation for at least 1 h before application. A normal mouse immunoglobulin G (IgG)a2 antibody (Santa Cruz Biotechnology) served as a negative control for p75 (at the same concentration as the primary antibody), and a normal rabbit IgG antibody (Santa Cruz Biotechnology) served as an additional negative control for TrkA (at the same concentration as the primary antibody). All slides were then rinsed. The slides for the NGF and p75 studies were incubated with biotinylated anti-rabbit, anti-mouse and anti-goat immunoglobulins in PBS containing carrier protein and 15 mM sodium azide (Link from DAKO LSAB+System, HRP, DAKO), and the slides for the TrkA study were incubated with labelled polymer, HRP anti-rabbit (from a DAKO EnVision+System, HRP-AEC anti rabbit-kit, DAKO).
After further rinsing, the sections for NGF and p75 were incubated with streptavidin conjugates to horse-radish peroxidase containing carrier protein and antimicrobial agents (Streptavidin HRP from DAKO LSAB+system, HRP, DAKO), followed by exposure to a diaminobenzidine urea H2O2 solution in distilled water (Sigma Fast tablets, Sigma) for 5 min. The sections for TrkA were exposed to AEC+substrate-chromogen (from a DAKO EnVision+System, HRP-AEC kit, DAKO) for 30 min. All sections were then counterstained with Mayer's haematoxylin (Pioneer Research Chemicals Ltd, Colchester, Essex, UK) (purple-blue staining). Brown staining indicated expression of either NGF and p75 and red-brown staining indicated expression of TrkA. Unless otherwise stated, all dilutions were performed with PBS (Biological Industries), which served also as the main rinsing solution, and the incubations were carried out at room temperature. Two sections per slice were stained for NGF, p75 and TrkA.
In order to demonstrate the number of follicles that underwent immunostaining, we conducted a computerized evaluation of follicular number per section (analySIS, Soft Imaging System, Digital Solutions for Imaging and Microscopy, System GmbH, Munster, Germany), and the follicles were classified as (Gougeon, 1996) primordial, primary, secondary (see definitions in the Introduction), or antral, i.e. follicles with a fluid-filled cavity (antrum) within the cuboidal GC and flat GC bordering the basement membrane.
RNA extraction
RNA samples were extracted from the same ovarian samples used for immunocytochemistry. The frozen ovarian fragments were partially thawed at 37°C. Thereafter, they were rapidly removed from the semi-frozen DMSO solution and placed in TRizol Reagent (Pioneer Research Chemicals Ltd) at room temperature and homogenized (Abir et al., 2004). To obtain RNA fractions (supernatants) from these homogenates, chloroform (Biolab, Jerusalem, Israel) was added first, and then a solution of phenol–chloroform–isoamyl alcohol (Sigma), followed by subsequent centrifugation at 4°C. To obtain RNA pellets, these RNA fractions were mixed with isopropanol (Biolab) and kept overnight at –20°C, followed by centrifugation at 4°C. Finally, in order to stabilize the mRNA pellet, ethanol (75%) was added. These samples were kept at –80°C until RT–PCR was performed.
RT–PCR
Frozen total RNA samples were centrifuged at 4°C, 13 000 g for 30 min. After the supernatant was completely removed, the pellets containing RNA were resuspended in 50 µl RNase free diethyl procarbonate-treated water. The concentration of each sample was measured by spectrophotometer (Cary UV 100, Varian, Mulgrave, Australia), and the sample was stored at –70°C. A total of 0.5 µg RNA was used for cDNA synthesis in the presence (RT+) or absence (RT–) of reverse transcriptase. The final reaction mixture containing RNA 10 µl, Oligo DT (dT) 1 µl, 10 mM dNTP 1 µl, 5x RT buffer 4 µl, 0.1 M dithiothreitol 2 µl, RNAsin 1 µl and 100 units of Moloney murine leukemia virus (M-MLV) reverse transcriptase (GIBCO BRL, Burlington, Ontario, Canada) was incubated at 37°C for 1 h. The reaction was terminated by heating at 95°C for 5 min. cDNA amplification was performed with two rounds of hemi-nested PCR primers to increase the specificity and yield of the PCR product. The total 50 µl PCR reaction contained 5 µl reverse-transcribed cDNA, 5 µl of 10x PCR buffer (QIAGEN, Ontario, Canada), 50 mM of each dNTP, 2.5 units of Taq polymerase and 0.4 µM of each primer. Each reaction was overlaid with 50 µl of mineral oil and heated to 95°C for 4 min. PCR was carried out essentially as previously described (Ao et al., 1994), for 25–29 cycles with outer primers and 30–35 cycles with inner, hemi-nested primers at the appropriate annealing temperatures for each set (Table III). For the hemi-nested PCR, 2 µl of primary product was added to 28 µl of freshly prepared mix, as above. The amplified products were subjected to 1% agarose gel electrophoresis using a 100 base pair (bp) ladder as a reference for fragment size and was then stained with ethidium bromide. The RT–PCR products of NGF, p75 and TrkA from human fetal and adult ovaries were purified with the QIAquick Purification Kit (QIAGEN) and sequenced commercially.
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Statistical analysis
The number of follicles in the immunocytochemically stained samples were analysed by
2-test and Fisher's exact test. The number of follicles, both total and by class (primordial, primary, secondary and antral), was compared between fetuses and adolescents/women, and differences in the number of primordial, primary, secondary and antral follicles were compared within each ovarian source (adolescents/women or fetuses). |
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Immunocytochemical staining for NGF, p75 and TrkA
Figures 1, 2 and 3 show the immunocytochemical staining for NGF, TrkA and p75, respectively. The positive staining intensities for NGF in all the ovarian cells varied from low to strong (Figure 1A and B). Fetal oocytes/oogonia showed full cytoplasmic NGF staining without any nuclear staining, with no differences between oocytes and oogonia (Figure 1A). By contrast, all the oocytes from women/adolescents exhibited partial cytoplasmic staining for NGF as well as nuclear staining (Figure 1B). NGF was also detected in GC of follicles from primordial stages onward, and in some stroma cells in all the fetal and adult/adolescent samples (Figure 1A and B). There was no relation between the staining pattern for NGF and follicular size or class, fetal or adult age, normal or abnormal status of the fetus, or specific fetal malformations, including chromosomal aberrations.
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TrkA staining was demonstrated mainly in oogonia/oocytes and in some stroma cells (Figure 2A and B). The positive staining intensities for TrkA in all the ovarian cells varied from low to moderate (Figure 2A and B). Most of the fetal oocytes/oogonia showed full cytoplasmic staining without nuclear staining (Figure 1A), although a minority had only partial cytoplasmic staining (Figure 1A). There were no differences in staining pattern between fetal oocytes and oogonia. In most of the ovaries from women/adolescents, there was partial cytoplasmic staining with nuclear staining, although a small portion of the follicles had full cytoplasmic staining (Figure 2B). Staining in the GC was identified only in some of the samples: 35% of the fetal samples and 50% of the samples from women/adolescents (Figure 2A and B). There was no relation between the staining pattern for TrkA in the ovarian cells and follicular size or class, fetal or adult age, normal or abnormal state of the fetus, or specific malformations, including chromosomal aberrations.
Positive staining for p75 was detected only in some stroma cells of fetuses aged 19–21 GW (Figure 3A), but not in ovaries of older fetuses or adults. There was no relation between the staining pattern for p75 in the stroma cells and the normality or abnormality of the fetuses, or the specific malformations including chromosomal aberrations. The negative controls did not stain for NGF, p75 or TrkA and were blue-purple (Figures 1C, 2C and 3B).
A total of 119 follicles were counted in the immunostained ovarian sections from the women and girls: 81.5% primordial, 3.4% secondary (4 follicles) and one antral. A total of 8006 follicles (P<0.0001, compared with women) were counted in the immunostained ovarian sections from fetuses (P<0.0001, compared with follicles from women): 95.7% primordial (7662 follicles, P<0.0001, compared with primordial follicles from women) and only 0.1% secondary (6 follicles). There was a significantly higher total number of primordial follicles than primary and secondary follicles (P<0.0001).
Detection of NGF, p75 and TrkA transcripts
The cDNA amplification primers for NGF, p75 and TrkA were designed to span introns so that genomic DNA contamination could be eliminated. Hemi-nested RT–PCR analysis was performed on ten adult and 34 fetal samples (Figure 4). The RT–PCR assay was repeated at least three times, and all samples yielded the expected fragment sizes (Table III; Figure 4). There was no difference in the pattern of expression of NGF and TrkA genes between the samples from women and fetuses. However, the transcripts for p75 were detected only in fetal ovaries aged less than 22 GW; because the RT–PCR analysis was not quantitative, the presence of differential transcript levels could not be distinguished among these genes or between the samples from women and fetuses. Constitutively expressed hypoxanthine phosphoribosyl transferase (HPRT) gene was used as the positive control for the RT reaction. There was no contamination of genomic DNA in any of the samples tested, and all negative controls (RT–) processed without reverse transcriptase yielded no amplification product. Sequence analysis of the RT–PCR products confirmed the identity of NGF, p75 and TrkA (data not shown). Sequences corresponding to positions 289–476, positions 1218–1369, as well as positions 962–1156 were identical to the human for NGF, p75 and TrkA gene-bank-published mRNA sequences, respectively (www.ncbi.nlm.nih.gov/).
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Discussion |
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The present study showed for the first time, the concurrent expression of NGF and its TrkA receptor in human adult and late-gestational fetal ovaries by both immunocytochemistry and RT–PCR. Immunocytochemical staining localized the protein for NGF in oocytes as well as GC of human fetal and adult primordial, primary and secondary ovarian follicles; TrkA was expressed mainly in oocytes and in 40% of the samples also in GC. The p75 receptor was identified only in some stroma cells in ovaries of fetuses aged less than 22 GW. Transcripts of NGF and TrkA were identified in all adult and fetal ovaries tested, and transcripts of p75 were detected in ovaries of fetuses aged less than 22 GW.
The range of cells that are responsive to NGF is narrower than that activated by other NTs (Chao and Hempstead, 1995). The interactions between NGF's two receptors, p75 and TrkA, are complex and form the specific cellular affinity for NGF (Chao and Hempstead, 1995). P75 receptors have a much wider distribution than any of the Trk receptors in various cell types including Schwann cells, motor cells, meningeal cells, dental pulp cells, hair follicle cells and cerebellar Purkinje cells. Moreover, during development, the expression of TrkA in the nervous system is limited to the sensory and sympathetic neurons in the peripheral nervous system and cholinergic neurons in the basal forebrain. Most NGF-responsive cells express both p75 and TrkA, though their expression of TrkA is usually limited. Due to the relatively higher levels of p75, the majority of NGF binding is to p75 receptors, with the TrkA receptors becoming low affinity. The rapid rates of p75 association and dissociation for NGF account for the low-affinity nature of the binding. TrkA displays very slow dissociation behaviour, suggesting a far greater affinity, but its unusually slow rate of NGF binding gives rise to a lower affinity for NGF. However, when p75 and TrkA are co-expressed, a high-affinity site is formed by the two receptors leading to a 25-fold increase in NGF binding compared with that of TrkA alone. Thus, changes in levels of p75 and TrkA can influence the overall binding affinities for NGF.
The sympathetic nerve fibres that innervate the rat ovary depend on NGF activation (Lara et al., 1990). NGF ovarian content is, in turn, regulated by ovarian innervation, such that denervation leads to NGF accumulation (Lara et al., 1990; Dissen et al., 1991). The presence of NGF mRNA and protein in ovaries of juvenile rats was indicated by the ability of the ovaries to both transcribe the NGF gene and translate its mRNA into the protein. An active p75 receptor as well as p75 mRNA were detected as well. Thus, the rat ovary seems to be a site for p75 synthesis.
More recent studies suggested that NTs such as NGF are involved not only in ovarian innervation but also in follicular assembly and initiation of folliculogenesis in rats and mice (Ojeda et al., 2000). This was supported by immunohistofluorescence-confocal microscopy studies and hybridization histochemisty studies, which revealed the expression of p75 and TrkA in ovaries of infantile mice during follicular assembly and initiation of folliculogenesis (Dissen et al., 2001).
Furthermore, in a study of the developing rat ovary (Dissen et al., 1995), researchers detected both NGF and TrkA mRNAs during late fetal development. Their abundance decreased post-natally at the time of follicular assembly. The authors suggested that this signalling complex, which is prominent in the fetal ovary but is markedly reduced during the time of follicular formation, may play a role in processes other than cellular differentiation (Ojeda et al., 2000).
Immunocytochemical studies reported that p75 is expressed in fetal ovarian mesenchymal cells in the rat. By the end of gestation, the pocket-like cellular structures expressing p75 had separated the epithelial pre-GC into small groups surrounding oocyte clusters (Dissen et al., 1995; Ojeda et al., 2000). This process culminated post-natally with the formation of primordial follicles. There was also a significant increase in p75 mRNA, which was maintained at the time of follicular assembly even though the receptor was not expressed in either GC or oocytes (Ojeda et al., 2000). These results relate very well with our detection of p75 RNA and protein only in ovaries of fetuses under age 22 GW, when human follicular assembly takes place.
Immunocytochemical analyses of ovaries from rhesus monkeys revealed the expression of p75 not only in neuron-related cells but also in non-neuronal endocrine cells, specifically theca cells (Dees et al., 1995). The expression of p75 mRNA also in the rhesus ovary indicated that the gland possesses the ability to synthesize this receptor.
Very little is known about the expression of NTs or their receptors in human ovaries. In RT–PCR studies, Anderson et al. (2002) detected the gene for TrkA in ovaries from fetuses aged 13–21 GW, and confirmed the expression of the p75 protein by immunocytochemical and immunoblotting studies. The protein for p75 was predominantly localized in ovarian stroma cells surrounding, but not intermixing with, the oogonia; there was no p75 staining in the GC (Anderson et al., 2002). Anesetti et al. (2001) used standard immunocytochemistry and immunohistofluorescence-confocal microscopy to investigate the expression of p75 in pathologically normal human ovaries from three spontaneously aborted fetuses aged 24–37 GW and in three post-natal ovaries from infants aged 13 days to 10 months who died of sudden death syndrome. A population of p75 immunoreactive cells with a neuronal appearance was identified immunocytochemically mostly in the medullar portion of the ovary among blood vessels, but also in the ovarian cortex, in fibres derived from extrinsic nerves located in the adventitia of blood vessels. Immunohistofluorescence-confocal microscopy localized p75 in some of the neuronal bodies. More importantly, theca cells of growing follicles were p75-positive, similar to the findings in rhesus monkey ovaries (Dees et al., 1995).
Our results are in line with those of Anderson et al. (2002). We detected the expression of NGF and TrkA in all the samples from the women, as well as in the samples from the fetuses, at both the mRNA and protein levels. At the same time, p75 was detected at both levels only in the ovaries from fetuses aged 19–21 GW—in the same age range studied by Anderson et al. (2002)—
but not in older fetuses or women. However, our samples from women were cortical, not medullary, so they lacked the ovarian portions reported by Anesetti et al. (2001) to express p75; in addition we removed all visible blood vessels from all the samples before the analyses. Another explanation for the differences in our results from the study of Anesetti et al. (2001) may be in the fetal samples. We studied a larger number of ovaries than the earlier study (Anesetti et al., 2001), and it is possible that our fetal samples were more viable, as the authors failed to mention if a uterine death preceded the spontaneous abortions. Similarly, the viability of their neonatal ovaries must be questioned, because the period between death and ovarian collection was not noted. In addition, Anesetti et al. (2001) studied fetal ovaries after follicular assembly and, therefore, did not identify p75 in stroma cells, as we did and was reported by Anderson et al. (2002). The lack of p75 expression in our ovarian samples from fetuses over 21 GW as well as from women/adolescents was confirmed independently by two laboratory methods, immunocytochemistry and RT–PCR, further supporting the accuracy of our data. By contrast, Anesetti et al. (2001) used only immunocytochemical techniques, and their negative controls for p75 were unconvincing because they consisted only of the omission of the primary and secondary antibodies, whereas ours included non-specific mouse antibodies. Moreover, Anesetti et al. (2001) conducted their immunocytochemical studies on frozen sections, while we used paraffin sections. It is well documented that only very few growing follicles are present in ovaries of human fetuses and young babies (Gosden, 1995; Gougeon, 1996). Therefore, it is very probable that the number of growing follicles in the study of Anesetti et al. (2001) was very limited, thereby precluding conclusive results regarding the expression of p75 in the theca cells.
Mice carrying a null mutation of the NGF gene were found to express p75 and TrkA in their ovaries by immunohistofluorescence-confocal microscopy and hybridization histochemistry studies (Dissen et al., 1995; Ojeda et al., 2000). At the end of the first post-natal week, the population of primary and secondary follicles was markedly reduced, and the number of primordial follicles was marginally reduced. Apparently, a deficiency in NGF disrupts the subsequent development of primordial follicles. The reduction in the rate of somatic mesenchymal cell proliferation before the formation of primordial follicles, detected both in vivo and in vitro, led to an increased number of oocytes that failed to be incorporated into follicular structures (Ojeda et al., 2000). More recent studies have shown that the levels of FSH-R mRNA were also reduced in these mice (Romero et al., 2002). These results (Ojeda et al., 2000; Dissen et al., 2001) are in line with earlier studies showing that immunoneutralization of NGF during rat neonatal life delayed follicular formation (Dissen et al., 1991). Thus, NGF might play a role in various stages of follicular development in rodents (Ojeda et al., 2000; Romero et al., 2002).
In contrast to NGF null mice, p75 mutant mice had a normal population of primordial, primary and secondary follicles by the end of the fifth post-natal day (Lee et al., 1992; Ojeda et al., 2000). Moreover, ovaries from p75 null mice showed an increased number of primary and secondary follicles (Ojeda et al., 2000). Thus, findings suggest that p75 may act as a modulatory signal in mesenchymal pre-thecal cells, restraining follicular development beyond the primordial stage.
One group cultured fetal and neonatal mouse ovaries with an inhibitor of the Trk receptors, which decreased the survival of germ cells in newly formed primordial follicles (Spears et al., 2003). This effect was reduced by the addition of basic fibroblast growth factor to the culture medium. When 2-day-old rat ovaries containing mostly primordial follicles were placed in organ culture and treated with NGF (Romero et al., 2002), FSH-R mRNA expression increased and the ovary acquired the capacity to respond to FSH with cAMP formation and growth to secondary stages. Thus, exposure to NGF leads to the formation of biologically active FSH-R. Accordingly, culture of human ovaries from five morphologically normal fetuses aged 13–16 GW with a Trk inhibitor reduced the number of surviving oogonia (Spears et al., 2003).
The expression of NGF and its receptors shown in the present study suggests that it might be involved in follicular assembly and in the activation of primordial follicles as shown in other mammals (Ojeda et al., 2000). However, in order to further elucidate this possibility, NGF should be added to cultured human primordial follicles. On the basis of our findings and those of others (Ojeda et al., 2000; Anderson et al., 2002) that p75 expression can be detected only during follicular assembly, we speculate that the role of p75 may be limited to this stage. Therefore, theoretically at least, the inclusion of NGF or other NTs in the culture medium of fetal ovaries before follicular assembly might enhance the formation of follicles in vitro. Moreover, as the two receptors for NGF were not identified in all GC, it is possible that growth factors, or NTs other than NGF are involved in the initiation of folliculogenesis. Further studies in laboratory animals as well as in humans are still needed to clarify the possible roles of various other growth factors on early folliculogenesis.
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Acknowledgements |
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The study was partially supported by a research grant from the Israel Cancer Association (R.A., B.F.). The authors are indebted to Ms G. Ganzach from the Editorial Board of Rabin Medical Center for the English editing, to the staff at the Gynecology Ward for their help in locating suitable patients; to the Ultrasound Unit for identifying fetal gender and to Dr S. Freimann from the Felsenstein Medical Research Center, Beilinson Campus for her assistance with the RNA extraction.
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Submitted on February 7, 2005; accepted on February 23, 2005.
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