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Medical Research Council Human Reproductive Sciences Unit, Center for Reproductive Biology, and Section of Biomedical Sciences (N.S.), University of Edinburgh, Edinburgh, United Kingdom EH3 9ET
Address all correspondence and requests for reprints to: Dr. R. A. Anderson, Medical Research Council Human Reproductive Sciences Unit, Center for Reproductive Biology, 37 Chalmers Street, Edinburgh, United Kingdom EH3 9ET. E-mail: r.a.anderson{at}hrsu.mrc.ac.uk
Abstract
Mammalian ovarian development is characterized by a sequential pattern of mitotic proliferation of oogonia, initiation then arrest of meiosis, and primordial follicle formation. The factors regulating these processes are poorly understood. The neurotropins are survival and differentiation factors in the nervous system, acting via high affinity receptors of the trk protooncogene family and the low affinity p75 nerve growth factor receptor, and have also been described in the rodent ovary, where changes in NT4/TrkB gene expression have been detected at the time of primordial follicle formation. There are no data on neurotropin expression in the normal human ovary. We have investigated the expression and localization of neurotropins and their receptors in the midtrimester human fetal ovary (13–21 wk gestation). Expression of mRNA for neurotropins and their receptors was detected by RT-PCR. Clusters of oogonia were found to be the predominant site of NT4 mRNA expression using in situ hybridization. However, at later gestations granulosa cells of primordial follicles showed increased expression, with lesser expression in the enclosed oocytes. NT4 protein was also localized to the granulosa cells by immunohistochemistry and at earlier developmental stages to epithelioid cells, which were mingled with clusters of oogonia not expressing NT4. TrkB receptor protein was localized by immunohistochemistry to germ cells at all gestations examined. The p75 nerve growth factor receptor protein was exclusively expressed in the ovarian stroma. These data demonstrate the expression of neurotropins and their receptors within the human fetal ovary. Developmental changes in the pattern of expression of NT4 around the time of primordial follicle formation suggest that neurotropins may be involved in signaling between somatic cells and germ cells at this crucial stage of ovarian development.
THE DEVELOPMENT of the mammalian ovary is initiated after migration of primordial germ cells from the extraembryonic mesoderm of the yolk sac to the nephrogonadoblastic ridge (1). The germ cells proliferate both during and after migration, and associate with mesonephric cells (2). This results in nests of syncitial germ cells surrounded by a meshwork of the mesonephric-derived ovarian stroma or cell streams (3). The germ cells subsequently enter meiosis only to arrest at diplotene of the first meiotic division and at this time point become surrounded by somatic cells, thus forming primordial follicles (4). This process, allowing communication between oocyte and somatic cell, is crucial for the survival of both cell types (5). As reproductive life span is determined by the continuing presence of primordial follicles, the regulation of germ cell proliferation and survival in the form of primordial follicles is central to reproductive potential. Waves of atresia of oogonia have been identified at particular stages of development (6, 7), but the factors and pathways involved are largely unknown, particularly in the human.
The neurotropins are a family of related growth factors that are of major importance in the regulation of neuronal survival and differentiation. Nerve growth factor (NGF) is the founder member (8) and several other neurotropins have been identified, including brain-derived neurotropic factor and neurotropins 3 and 4 (NT3 and NT4, the latter also called NT5) (9). The biological actions of the neurotropins are mediated by tyrosine kinase receptors encoded by the trk protooncogene family, known as TrkA, TrkB, and TrkC (10). Truncated isoforms of Trk receptors lacking the intracellular tyrosine kinase domain have also been described (10, 11, 12). In addition, all neurotropins are recognized by a more widely expressed low affinity receptor known as p75 nerve growth factor receptor (NGFR), which is a member of the tumor necrosis receptor superfamily (13). Neurotropins may also have roles in nonneuronal systems (14, 15), and several members of the neurotropin family and their receptors have been demonstrated in the ovary (16, 17, 18, 19, 20, 21, 22). NT4 appears to be expressed by oocytes in both the rat and Xenopus (20, 22), and the expression of NT4 and its cognate high affinity receptor TrkB, but not other neurotropins/receptors, was found to be increased at the time of primordial follicle formation in the neonatal rat ovary (22). Conversely, expression of NGF and its high affinity receptor Trk A were reduced at this time. Follicular development was reduced in a NGF-null mutant model (23), although the number of primordial follicles was maintained. A specific and marked increase in NGF and TrkA receptor expression has also been demonstrated at the time of ovulation and suggested to mediate thecal cell differentiation at that time (24, 25).
There is therefore increasing evidence that the various neurotropins may have specific roles in ovarian development and function, but no data are currently available on their presence in the normal human ovary. We have therefore investigated the presence and distribution of the neurotropins and their receptors in the developing human ovary during the period leading up to the formation of primordial follicles.
Materials and Methods
Tissue samples
Human fetal ovaries were obtained after medical termination of pregnancy. Women gave written consent according to national guidelines (26), and the study was approved by the Lothian Pediatrics/Reproductive Medicine research ethics subcommittee. Termination of pregnancy was induced by treatment with mifepristone (200 mg, orally), followed by PGE1 analog (Gemeprost, Beacon Pharmaceutical, Tunbridge Wells, UK; 1 mg every 3 h, per vaginam). None of the terminations was for reasons of fetal abnormality, and all fetuses appeared morphologically normal. Gestational age was determined by ultrasound examination before termination and was confirmed by subsequent direct measurement of foot length. A total of 20 specimens were used for this study.
Ovaries were dissected free and either fixed for immunohistochemical analysis or snap-frozen and stored at -70 C. Fixation was carried out in Bouins for 5 h, followed by transfer to 70% ethanol before processing into paraffin using standard methods.
Isolation of RNA and synthesis and amplification of cDNA
Total RNA was extracted from snap-frozen samples of fetal ovary (13–21 wk) using the RNeasy mini kit (QIAGEN, Crawley, UK). RNA was treated with deoxyribonuclease (Life Technologies, Inc., Paisley, UK), RT was performed using a first strand cDNA synthesis kit (Roche, Lewes, UK), and PCR was performed as previously described (27). Briefly, 1 µg total RNA was incubated with oligo(deoxyribonucleic)18 primer for 10 min at 65 C and then placed on ice. A reaction mix comprising buffer, 1 mM each of dNTP, ribonuclease inhibitor, and 50 U 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. Subsequently, PCR was performed by incubating 1-µl cDNA samples with Taq DNA polymerase (AGS Gold, Hybaid, Ashford, UK) in buffer with 0.2 mM 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. Positive control samples (rat brain) were also run in parallel (not shown). PCR amplification was carried out for 35 cycles. Primers specific for human neurotropins and their receptors were used (Table 1
), designed to span an intron to ensure that genomic DNA was not amplified. Primers for the constitutively expressed gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used to confirm the integrity of the RNA and the efficacy of the PCR reaction. The identities of all PCR products were confirmed by direct sequencing using a PE Applied Biosystems 373A automated sequencer (Foster City, CA).
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Riboprobes were generated using a PCR strategy to incorporate SP6 or T7 phage promoter sequences into NT4 PCR product. Briefly, for the antisense probe, the NT4 PCR product was amplified using NT4 forward primer (5'-CTTTCGGGAGTCAGCAGGTGC-3') and NT4 reverse primer, which had the T7 promoter sequence added to its 5'-end (5'-TAATACGACTCACTATAGGGAGACAGGCAGTGTCAATTCGAATCC-3'). For the sense probe, product was amplified using the NT4 forward primer linked to the SP6 promoter sequence (5'-ATTTAGGTGACACTATAGAAGTGCTTTCGGGAGTCAGCAGGTGC-3') and NT4 reverse primer (5'-CAGGCAGTGTCAATTCGAATCC-3'). pGEM-T plasmid DNA containing human NT4 insert (nucleotides 401-1099) was used as a template. Primers specific to human NT4 were used to make the plasmid insert (Table 1
). After PCR, products were purified (High Pure PCR product purification kit, Roche) and used directly for transcription with T7 or SP6 RNA polymerase to generate antisense and sense riboprobes, respectively. Probes were labeled with digoxigenin using a commercially available kit (Maxiscript, Ambion, Inc., Huntingdon, UK), incubated with ribonuclease-free deoxyribonuclease I and purified through Chromaspin columns (DEPC-100, CLONTECH Laboratories, Inc., Palo Alto, CA).
Sections (5 µm) were cut over diethylpyrocarbonate-treated water and transferred to SuperFrost Plus-coated slides (BDM, Poole, UK), dewaxed in xylene, and rehydrated through a graded series of ethanol. Slides were then treated with 1.5 µg/ml proteinase K at 37 C for 10 min. Sections were incubated with prehybridization buffer, then hybridized at 50 C overnight with hybridization buffer containing 2 µl appropriate riboprobe/50 µl hybridization buffer. Sections were then incubated in ribonuclease A and blocked with avidin and biotin (Vector Laboratories, Inc., Peterborough, UK) before detection of digoxigenin label using sheep antidigoxigenin (1:100 in normal rat serum/Tris-buffered saline, Roche), biotinylated rabbit antisheep IgG (1:500 in normal rat serum/Tris-buffered saline, Vector Laboratories, Inc.), avidin-biotin horseradish peroxidase-linked complex (DAKO Corp., Copehagen, Denmark), and diaminobenzidene liquid substrate-chromagen system (DAKO Corp.). Sections were then counterstained in hematoxylin, dehydrated, and mounted with DPX.
Immunohistochemistry
Immunohistochemistry was performed as previously described (27). Sections (5 µm) were mounted on TESPA (Sigma, Poole, UK)-coated slides, dewaxed, and rehydrated. After inhibition of endogenous peroxidase activity by incubation in 3% H2O2 in methanol, sections were blocked in normal swine, donkey, or rabbit serum (Diagnostics Scotland, Carluke, UK) for NT4, TrkB, and p75 NGFR detection, respectively, and with avidin and biotin (both from Vector Laboratories, Inc.). The following primary antibodies were used: NT4 (rabbit polyclonal, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), TrkB (chicken polyclonal, Promega Corp., Southampton, UK), p75 NGFR (mouse monoclonal, Neomarkers, Freemont, CA). These were applied at dilutions of 1:100, 1:25, and 1:25, respectively, in the appropriate serum at 4 C overnight. Sections were then washed and incubated with appropriate biotinylated secondary antibody diluted 1:500 (NT4 and p75 NGFR, Diagnostics Scotland, TrkB, The Jackson Laboratory, Bar Harbor, ME). Sections were incubated with avidin-biotin horseradish peroxidase-linked complex (DAKO Corp.) according to the manufacturer’s instructions. Bound antibody was visualized using 3,3'-diaminobenzidine tetrahydrochloride (DAKO Corp.). Primary antibodies were omitted as negative controls.
All sections were counterstained with hematoxylin, dehydrated, mounted, and visualized by light microscopy. Images were captured using an Provis microscope (Olympus Corp., London, UK) equipped with a Kodak DCS330 camera (Eastman Kodak Co., Rochester, NY) and assembled using Photoshop 5 (Adobe, Becton Dickinson, Inc., Mountain View, CA).
Immunoblotting
Fetal ovaries were homogenized in denaturing buffer containing 1% SDS. Samples (20 µg protein) were diluted with an equal volume of reducing loading buffer [187 mM Tris (pH 6.8), 2% SDS, 2% ß-mercaptoethanol, 1% sucrose, and 0.01% bromophenol blue] and boiled for 5 min. Proteins were separated by SDS-PAGE on a 4–20% gradient Tris-glycine gel (Novex, Invitrogen, Paisley, UK) in parallel with prestained protein mol wt markers (Bio-Rad Laboratories, Inc., Richmond, CA) and blotted onto polyvinyldifluoride membranes (Amersham Pharmacia Biotech, Little Chalfont, UK). Membranes were blocked in 3% (wt/vol) BSA (Sigma) and 5% powdered milk and then incubated overnight with the primary antibody. Antibodies to NT4 (rabbit polyclonal, Santa Cruz Biotechnology, Inc.), full-length TrkB (rabbit polyclonal, Oncogene, Cambridge, MA), and p75 NGFR (mouse monoclonal, Neomarkers) were used at dilutions of 1:500, 1:50, and 1:500, respectively. Primary antibody was omitted as a negative control. Bound antibody was detected using horseradish peroxidase-linked secondary antibodies (1:4000; Amersham Pharmacia Biotech) and the enhanced chemiluminescence visualization system (Amersham Pharmacia Biotech) according to the manufacturer’s instructions.
Results
RT-PCR
After RT-PCR, cDNA was amplified from RNA extracted from fetal ovaries for each of the neurotropins, NGF, NT3, NT4, and brain-derived neurotropic factor (Fig. 1). cDNA for each of the receptors, TrkA, full-length TrkB, the truncated form of TrkB, TrkC and p75 NGFR, were also identified (Fig. 1). Positive results were found at all gestations examined for each of these neurotropins and their receptors over the range 13–21 wk. Products of 228 and 204 bp were detected for TrkC (Fig. 1D), representing spliced variants of the gene (28), both confirmed by direct sequencing. In some samples an additional PCR product of 300 bp was detected; sequence analysis showed that it was unrelated to the tyrosine kinase receptor family.
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Hybridization histochemistry demonstrated that the germ cells were the predominant site of expression of NT4 mRNA within the ovary before the formation of primordial follicles (Fig. 2, A and B). The pattern of expression clearly demarcated the ovarian stroma, within which some cells showed NT4 mRNA expression, from the more uniformly and intensely stained clusters of oogonia (Fig. 2A). No marked change in the level or pattern of expression was detected before the formation of primordial follicles. However, at that stage of development, intense staining was detected in the flattened granulosa cells surrounding the enlarged oocytes (Fig. 2C), in which NT4 expression was reduced, but still present. Less mature oogonia, situated more peripherally in the ovary, continued to express NT4 mRNA as in the earlier gestations examined. The cells of the ovarian surface epithelium did not express NT4 mRNA.
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Expression of NT4, TrkB, and p75 NGFR proteins was detected by immunohistochemistry in all specimens examined across the gestational range 13–21 wk. Oogonia showed weak cytoplasmic immunostaining for NT4 at all gestations (Fig. 2, D–F), but marked staining was detected in epithelioid cells among and immediately surrounding the nests of germ cells, consistent with early differentiation of pregranulosa cells (Fig. 2D). This pattern strikingly outlined the germ cells, both individually and in clusters. Generally, cells of the ovarian stroma showed only slight staining, although individual cells at the edge of the stroma, thus in close proximity to oogonial clusters, showed more marked staining (arrow, Fig. 2D). The most intense expression of NT4 protein was, however, seen in the cytoplasm of flattened granulosa cells of primordial follicles at later gestations. This pattern of staining very clearly demarcated formed primordial follicles predominantly located in the medullary region of the ovary from the more peripheral oogonia; thus, a medullary/cortical gradient was observed (Fig. 2, E and F). The pattern of distribution of NT4 protein therefore differed from that of NT4 mRNA at earlier gestations, but was similar once primordial follicles were formed. No staining of the ovarian surface epithelium was detected. Using an antibody specific to full-length TrkB, the protein was immunolocalized to the cytoplasm of ovarian germ cells across the gestational range examined (Fig. 2, G and H). In particular, clear staining of the cytoplasm of oocytes in primordial follicles was observed (Fig. 2H), with faint staining of the cytoplasm of pregranulosa cells.
The distribution of expression of p75 NGFR protein was very different. p75 NGFR was predominantly localized to cells of the ovarian stroma at all gestations examined (Fig. 2, I–K), thus clearly demonstrating the branching pattern of the stroma from the medulla toward the surface of the ovary, surrounding, but not becoming intermixed with, the clusters of oogonia. p75 NGFR immunostaining was not detected in germ cells at any gestation examined. In contrast to the expression of NT4, the flattened granulosa cells of primordial follicles clearly did not express p75 NGFR, although it was present in the immediately adjacent cells of the ovarian stroma (Fig. 2K). p75 NGFR was not expressed by the ovarian surface epithelium.
Immunoblotting
The presence of NT4, TrkB, and p75 NGFR proteins in the fetal ovary was confirmed by immunoblotting. NT4 protein expression was detected as a single band of 21 kDa (Fig. 3A), also observed in the positive control (rat cerebral cortex). Two immunoreactive bands of 110 and 95 kDa were observed for TrkB (Fig. 3B). As the antibody specifically detects full-length TrkB, these bands may represent variously glycosylated forms of the full-length molecule (11) rather than the truncated form. A band of 110 kDa was also detected in the positive control of rat cerebral cortex. Differences in lower mol wt forms may reflect differences in the pattern of glycosylation between the two tissues (Fig. 3B). A prominent 75-kDa band representing the p75 NGFR protein was observed (Fig. 3C). An additional 65-kDa band was also detected, representing another form of the molecule as detected by others (29, 30). A similar band was detected in the positive control tissue. The intensity of expression was comparable among the samples used (13–21 wk gestation) for the three proteins, although insufficient samples were available for rigorous quantitative analysis. A negative control was also performed for all three proteins by omitting the primary antibody, and in all cases immunoreactivity was abolished (data not shown).
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The present results demonstrate the expression of neurotropin mRNA and protein within the developing human ovary. Expression of both the high affinity Trk receptors and the low affinity nonselective p75 NGFR receptor was also demonstrated. Localization of NT4 mRNA confirmed that the germ cells are the predominant site of expression of this neurotropin before the formation of primordial follicles. Expression was, however, developmentally regulated, thus after the formation of primordial follicles, the predominant site of expression of NT4 mRNA and protein was the flattened granulosa cell, whereas its cognate high affinity receptor TrkB was localized to the oocyte using immunohistochemistry. These data therefore indicate the involvement of neurotropins in this crucial step in ovarian development, i.e. in the formation of the essential structures of the ovary.
Tissue specimens were obtained after medical termination of pregnancy, induced by administration of the antigestogen mifepristone and PGE1. It is possible that the administration of these drugs, directly or indirectly, might have affected the expression of neurotropins in the fetal ovary, in which there is some expression of steroidogenic enzymes at these gestations (31). Hypoxia has also been demonstrated to modify expression of neurotropins and their receptors in several tissues (32, 33). Although the results presented here are in broad agreement with the limited data obtained in the developing rodent ovary (see below), it would be appropriate to corroborate the present data in specimens obtained by other methods.
Neurotropins, specifically NGF, were originally identified on the basis of their role in the regulation of neuronal survival (8). They are small, secreted proteins related to the TGFß superfamily. The neurotropin hypothesis holds that developing axons require target-derived factors for their survival, and that these factors are produced in limited quantity, resulting in competition between in-growing axons and selective survival (34). Trk-mediated cell survival requires protein synthesis, and neurotropins are predominantly believed to act by suppression of apoptosis. Neurotropins may also regulate cell migration (35) and differentiation (15). In addition to these prodevelopment roles, neurotropins can promote cell death. This had been suggested to be a function of the p75 NGFR receptor when present without the coexistence of Trk receptors, whereas in their presence p75 NGFR promotes cell survival (36). Differential effects of p75 NGFR activation on Trk receptor autophosphorylation dependent on both ligand and Trk subtype have been previously described (37). There is thus the potential for complex interplay between the different receptor pathways.
There are no previous data on the presence of neurotropins in the human ovary, but their expression has been demonstrated in the rat ovary (21, 22, 23). The immediate postnatal period in the rat is the time of primordial follicle formation, and increased expression of NT4 and its high affinity receptor TrkB, but not other neurotropins/Trk receptors, was demonstrated at that time (22). NT4 expression has been localized to the germ cell in both rodent and Xenopus (20, 22). The present results are therefore in broad agreement with the limited previous data in other species. NT4 protein was not, however, predominantly localized to the germ cells. The main site of localization was granulosa cells surrounding oocytes in primordial follicles and, at earlier developmental stages, in epithelioid stromal cells within clusters of oogonia. It appears likely that these cells are the precursors of the granulosa cells. This is similar to the pattern of development demonstrated in the rodent, where these cells are derived from the cells of the rete ovarii, which, in turn, derive from the mesonephros (reviewed in Ref. 2). The rete ovarii is also recognized to be of central importance in the regulation of the onset of meiosis (38) and of the cortical/medullary pattern in the rodent ovary (39, 40). The neurotropins may therefore be involved in the regulation of development of the oocyte in the human ovary at a number of levels.
A central component of the mechanism of action of neurotropins in the nervous system is that they are produced and released by the target cell in proportion to the final innervation density, taken up by the innervating neuron, and transported to the nucleus where they mediate their pro-survival effects (41, 42). The predominant localization of NT4 mRNA and protein in different cell types before primordial follicle formation is consistent with a similar pattern of secretion/uptake by germ cells and somatic cells, respectively, suggesting that NT4 is involved in the intercellular communication between these cells types. The change in pattern of expression of NT4 mRNA, being low in the oocyte and high in the granulosa cell after primordial follicle formation, indicates that the signals mediated by this pathway may vary according to the developmental status of the germ cell/somatic cell. The localization of TrkB to the germ cells suggests that these are a major site of NT4 action, whereas p75 NGFR was localized to the ovarian stroma and was not present in the granulosa cells of primordial follicles. The distribution of p75 NGFR in the human fetal ovary is therefore very similar to that in the neonatal rat ovary (22). The differential distribution of the several receptors may contribute to the regulation of neurotropin signaling in the immediate environment of the forming primordial follicle. The localization of other Trk receptors has not been investigated. In the newborn rat, TrkB mRNA appeared to be localized to presumptive pregranulosa cells (22). Whether this is a species difference or reflects a particular developmental stage is unclear at present. Developmental changes in the requirement for specific neurotropins during neuronal differentiation and survival have been described (34). It has long been recognized that oocytes must interact with somatic cells to form primordial follicles and survive (5). The intermingling of NT4-immunopositive epithelioid cells within the clusters of oogonia may indicate a crucial role for oogonial-derived neurotropins in determining germ cell survival by enhancing the movement and differentiation of somatic cells and thus promoting primordial follicle development.
Three Trk receptors with high affinity for specific neurotropins have been identified as well as the low affinity p75 NGFR receptor (10, 13). Truncated isoforms of TrkB and TrkC resulting from alternative splicing have also been described (10, 11, 12). The present results demonstrate that mRNA for both full-length and truncated isoforms of TrkB is expressed in the human fetal ovary. The truncated isoforms of the Trk receptors lack the intracellular, tyrosine kinase domain, but retain the ligand-binding domain, and may be found in both neuronal and nonneuronal cell types, although the truncated form may be more abundant in the latter (11, 43). Changing patterns of expression of full-length vs. truncated forms have been demonstrated during tissue repair in certain regions of the nervous system (44). TrkB-truncated isoforms have recently been suggested to be involved in neurotropin endocytosis (45) and in regulation of specific patterns of dendritic growth distinct from those mediated by the full-length isoform (46). Conversely, several neurotropins and their receptors may require to be coexpressed by specific cell types (47, 48). The relevance of Trk splice variants to ovarian development and function remains to be investigated.
Although the present study does not comprehensively localize within the ovary all identified members of the neurotropin family and their receptors, the differential localization of TrkB, p75 NGFR, and NT4 may indicate multiple roles for neurotropin signaling. Indeed, neurotropins have been implicated in several processes in the rodent ovary. Increased NT4/TrkB mRNA expression was associated with primordial follicle formation (22), whereas increased NGF/TrkA expression was induced by the LH surge during first ovulation (24) and is involved in the regulation of intercellular gap junction integrity between thecal cells (25). Increased NGF production within the ovary resulted in disruption of estrous cyclicity (49). It therefore appears that individual neurotropin/receptor pathways are involved in the regulation of specific intraovarian processes. Direct evidence for the importance of neurotropins in ovarian development is scanty. In vitro incubation of neonatal rat ovary with the nonselective Trk receptor antagonist K-252a reduced the number of primordial follicles present (22). Recent studies of mice with an inactivating mutation of TrkB are consistent with this pathway having an important role in primordial follicle formation (50), although transgenic NT4 knockout mice appear to be normally fertile (51). The ovaries of neonatal NGF-knockout mice also show reduced proliferation of mesenchymal cells and reduced primordial follicle growth, suggesting that both cell populations are targets for NGF action (23). Mice carrying a null mutation of the p75 NGFR gene appear to have normal numbers of ovarian follicles, but this receptor may be involved in the reduction in mesenchymal cell proliferation found in the NGF-knockout mouse (23), as this is the site of expression of the p75NGFR. Later stages of ovarian development in neurotropin/Trk knockout models have not been studied in detail, as they are generally nonviable (52).
These results therefore demonstrate the presence of neurotropins and their receptors in the developing human ovary. Differential patterns of expression between cell types and developmental changes, particularly associated with primordial follicle formation, suggest multiple roles for them in the regulation of germ cell and somatic cell proliferation, survival, and differentiation.
Footnotes
Abbreviations: GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; NGF, nerve growth factor; NGFR, nerve growth factor receptor; NT, neurotropin.
Received June 22, 2001.
Accepted October 29, 2001.
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