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Neurotrophin-4/5 and Neurotrophin-3 Are Present within the Human Ovarian Follicle but Appear to Have Different Paracrine/Autocrine Functions

Department of Obstetrics, Gynecology, and Reproductive Services (D.B.S., B.F., R.M.S., S.C.), Division of Reproductive Endocrinology and Infertility, University of Medicine and Dentistry of New Jersey (UMDNJ)–Robert Wood Johnson Medical School, New Brunswick, New Jersey 08901; and Department of Neuroscience and Cell Biology (C.F.D.), UMDNJ–Robert Wood Johnson Medical School, Piscataway, New Jersey 08854-5635

Address all correspondence and requests for reprints to: David B. Seifer, M.D., University of Medicine and Dentistry of New Jersey–Robert Wood Johnson Medical School, 303 George Street, Suite 250, New Brunswick, New Jersey 08901. E-mail: seiferdb{at}umdnj.edu.

Abstract

Neurotrophins are a family of soluble polypeptide growth factors widely recognized for their role in the mammalian nervous system. We first reported the unique presence of one neurotrophin, brain-derived neurotrophic factor (BDNF), in the follicular fluid of the human ovarian follicle. The BDNF receptor, Trk B, was identified in mouse oocytes, and BDNF accelerated first polar body extrusion in vitro. In the present study, we examined human follicular fluid and mouse immature oocytes to determine whether another Trk B ligand, neurotrophin-4/5 (NT-4/5), is present within the ovarian follicle and if so, whether it demonstrates activity similar to that of BDNF. We also examined whether a non-Trk B neurotrophin ligand, neurotrophin-3 (NT-3), is present within the follicle and has a possible role in oocyte maturation.

NT-4/5 and NT-3 were noted to be present in all human follicular fluid samples aspirated from follicles of women undergoing in vitro fertilization. NT-4/5, but not NT-3, significantly promoted mouse oocyte polar body extrusion. Trk C receptors were not noted to be present in mouse oocytes. This study demonstrates for the first time that NT-4/5 and NT-3 are present in the follicular fluid of the human ovary. These data suggest that NT-4/5, like BDNF, promotes oocyte nuclear maturation. In contrast, NT-3 does not promote oocyte maturation but may contribute to follicle-oocyte maturation by mechanisms not yet identified.

NEUROTROPHINS ARE A FAMILY of soluble polypeptide growth factors widely recognized for their roles in the mammalian nervous system. They include brain- derived neurotrophic factor (BDNF), neurotrophin-4/5 (NT-4/5), neurotrophin-3 (NT-3), and nerve growth factor (NGF). Although originally discovered in the nervous system, many members of the neurotrophin family are expressed in a variety of nonneuronal systems including the cardiovascular, immune, endocrine, and reproductive systems (1, 2).

BDNF, NT-4/5, NT-3, and NGF and their respective receptor tyrosine kinases (Trk B for BDNF and NT-4/5, Trk C for NT-3, and Trk A for NGF) have been shown to be expressed in the mammalian ovary (3), and several of these neurotrophins and their receptors have been demonstrated to play a role in rodent ovarian function such as ovulation (4, 5), steroid secretion (6), and follicular development (7, 8). Until recently, whether neurotrophins were present and serve similar roles in human ovarian function had not been addressed.

We first reported the presence of one such unique neurotrophin, BDNF, in the follicular fluid of the human ovarian follicle. We demonstrated its secretion by human cumulus granulosa cells and its promotion of mouse oocyte maturation presumably via its receptor, Trk B (9). In the present study, we examined whether another Trk B ligand, NT-4/5, may be found within the ovarian follicle and serve a functional role similar to BDNF. We also examined whether NT-3, a typically non-Trk B ligand, may be present with its high-affinity receptor, Trk C, within the ovarian follicle and serve a possible role in oocyte maturation.

Subjects and Methods

Human studies

Population. Twenty women who were younger than 40 yr of age (mean ± SE, 36.7 ± 0.6 yr) had follicular fluid containing granulosa cells aspirated while undergoing oocyte retrieval for in vitro fertilization (IVF). All had received leuprolide acetate (Lupron, TAP Pharmaceuticals, Inc., North Chicago, IL) for pituitary desensitization. After adequate suppression, two to eight ampules of gonadotropins were given daily in divided doses between morning and evening as previously described (10). Transvaginal follicular aspiration was performed under sedation 36 h after human chorionic gonadotropin injection. Approval of this study was obtained from the Institutional Review Board at the University of Medicine and Dentistry of New Jersey–Robert Wood Johnson Medical School.

Follicular fluid collection study. Follicular fluid and granulosa cells were isolated by aspiration of 23 follicles from 20 women undergoing IVF/embryo transfer. Clear follicular fluid without blood or flushing solution was obtained from one or more individual follicles measuring greater than 17 mm in diameter. Fluid was centrifuged at 1000 rpm for 10 min to remove the cellular component. The clear supernatant fraction was stored at -80 C for assay of NT-4/5 and NT-3.

Assays. NT-4/5 and NT-3 were determined using the commercially available, NT-4/5 Emax and NT-3 Emax immunoassay systems (Promega Corp., Madison, WI). The ELISAs were performed according to the manufacturer’s protocol. Follicular fluid was added to a 96-well immunoplate precoated with human NT-4/5 or NT-3 specific polyclonal antibodies. Plates were incubated at room temperature for 6 h with shaking. Antihuman-NT-4/5 or antihuman NT-3 and monoclonal antibody (reporter antibody) were used. After washing, the amount of specifically bound reporter antibody was detected using a species-specific antimouse-IgG antibody conjugated to horseradish peroxidase as a tertiary reactant. Unbound conjugate was removed by washing followed by incubation with a chromogenic substrate. Absorbency was measured at 450 nm using a microplate reader (Model Vmax kinetic microplate reader, Molecular Devices, Sunnyvale, CA). All samples were assayed in duplicate.

Anti NT-4/5 cross-reactivity with related neurotrophin factors (BDNF, NGF and NT-3) was reported to be less than 3% at 100 ng/ml. The detection sensitivity of the NT-4/5 ELISA was reported to be 9.4 pg/ml with an intra-assay coefficient of variation of 3.1% at a mean concentration of 229.7 pg/ml NT-4/5, according to the manufacturer. Our interassay coefficient of variation for the NT-4/5 ELISA was 5.1%.

Anti NT-3 was reported by the manufacturer to demonstrate less than 3% cross-reactivity with other related neurotrophin factors. The detection sensitivity of the ELISA is 10 pg/ml with an intra-assay coefficient of variation of 1.5% at mean concentration of 227.0 pg/ml NT-3, according to the manufacturer. The interassay coefficient of variation in our ELISA was 6.2%.

Several negative controls were included in each assay to rule out nonspecific binding of antihuman NT-4/5, NT-3 antibody, or anti-IgY binding to human serum IgG. Negative controls included: 1) omitting NT-4/5 or NT-3 during initial reaction with immobilized antibody, 2) absence of antibody specific to NT-4/5 or NT-3, and 3) absence of the reporting antibody. These negative controls were included in each assay performed to demonstrate the absence of nonspecific binding.

Mouse studies

Effect of NT-4/5 and NT-3 on in vitro maturation of mouse oocytes. Eighteen 4- to 5-wk-old female C57BL/6 mice (Taconic Farms, Inc., Germantown, NY) were given a single ip injection of 7.5 IU pregnant mare serum gonadotrophin (Sigma, St. Louis, MO). A total of 536 immature oocytes were harvested 48 h after gonadotropin injection. Cumulus cells were removed mechanically (27-gauge insulin needles) at room temperature. Immature oocytes were divided into control (n = 218) and experimental (n = 318) groups and cultured in 5% CO₂ in air, 37 C in human tubal fluid for 24 and 48 h with 1 ng/ml (n = 59), 10 ng/ml (n = 98), and 100 ng/ml (n = 41) of NT-4/5 (Peprotech, Inc., Rocky Hill, NJ) or 10 ng/ml (n = 60) and 100 ng/ml (n = 60) of NT-3 (Peprotech, Inc.) to examine the percentage of germinal vesicle breakdown (GVBD) and/or polar body extrusion. Control oocytes (n = 218) were cultured in human tubal fluid under the same conditions without addition of NT-4/5 (n = 158) or NT-3 (n = 60). The percentage of oocytes in a cohort of oocytes that demonstrated GVBD and/or first polar body extrusion was recorded after 24 and 48 h in culture.

Trk C receptor identification in mouse oocytes. Six mice (C57BL/6 mice, Taconic Farms, Inc.), 4–5 wk old, were injected ip with 6–8 IU pregnant mare serum gonadotropin (Sigma). Forty-eight hours later, mice were euthanized with CO₂, and ovaries were removed and placed in ice-cold PBS. Oocytes were isolated using 27-gauge needles under the stereoscope. Isolated oocytes were pooled into cold PBS until dissection was completed. Pooled oocytes were transferred into 4-well Nunc (Nunculon 176740, Nalge Nunc International, Rochester, NY) chambers in PBS. After replacing the PBS with 4% paraformaldehyde, wells were sealed with Parafilm and stored overnight at 4–8 C. After fixation, oocytes were washed three times with cold PBS and stored in PBS at 4–8 C until stained.

Mouse oocyte Trk C receptor staining was performed at room temperature using a modification of the procedure previously described for Trk B staining (3). In brief, oocytes (n = 152) were treated with blocking serum (15% goat serum with 0.1% Triton-X). Anti-Trk C-treated oocytes (n = 68) were then incubated 30–60 min at room temperature in 1:200 rabbit anti-Trk C antibody (sc-117, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) directed against a peptide mapping near the carboxy terminus of Trk C gp140. After incubation with Trk C antibody, oocytes were incubated 1 h at room temperature in goat antirabbit IgG (1:200) and then for 30–60 min in avidin-biotinylated enzyme complex (ABC reagent, Vectastain Elite, Vector Laboratories, Inc., Burlingame, CA). Between each oocyte treatment, oocytes were rinsed three times in PBS. The final buffer was replaced with diaminobenzidine tetrahydrochloride under stereoscopic visualization, with stain intensity recorded 10 min after addition of diaminobenzidine tetrahydrochloride. Control oocytes (n = 84) were treated as above except that rabbit anti-Trk C antiserum was omitted or anti-Trk C antibody was preadsorbed with Trk C peptide mimicking the receptor peptide sequence against which the antibody was directed. Positive controls consisted of sympathetic ganglia dissected from 1-d-old rat pups, fixed 2 h in 4% paraformaldehyde, then equilibrated in 30% sucrose before cryostat sectioning and mounting on glass slides. Ganglia were incubated with either anti-Trk C (1:200) antiserum or anti-Trk C antiserum adsorbed with blocking peptide (sc-117 P, Santa Cruz Biotechnolgy, Inc.).

Statistical analysis. Statistical analysis of data were performed using an unequal variance two-tailed t test, Fisher’s exact test, or ² for group comparisons where appropriate. Statistical significance was assumed at P value less than 0.05.

Results

NT-4/5 and NT-3 were noted to be present in all follicular fluid samples. The mean ± SE follicular fluid concentrations of NT-4/5 and NT-3 were 397.0 ± 71 pg/ml and 509.0 ± 97 pg/ml, respectively. NT-4/5 significantly influenced mouse oocyte polar body extrusion at 24 h with 10 ng/ml (<0.01) and at 48 h with 1.0 ng/ml (P < 0.05) and 10 ng/ml (P < 0.04; Fig. 1). NT-4/5 did not influence GVBD.

View larger version (20K): [in this window] [in a new window]   Figure 1. NT-4/5 influence upon mouse oocyte polar body extrusion. Oocytes from 18 4- to 5-wk-old mice (C57BL) were cultured for 24 and 48 h. Oocytes were divided into groups: control (n = 158); NT-4/5, 1 ng/ml (n = 59); NT-4/5, 10 ng/ml (n = 98); and NT-4/5, 100 ng/ml (n = 41). *, P < 0.01 (24 h, 10 ng/ml); **, P = 0.05 (48 h, 1 ng/ml); ***, P < 0.04 (48 h, 10 ng/ml).

Discussion

Within the last year, two members of the neurotrophin family have been identified within the human ovary (9, 11). BDNF was first demonstrated to be secreted by cumulus granulosa cells retrieved from women undergoing IVF and grown in culture (9). NT-4/5 was demonstrated to be present in the granulosa cells of human fetal ovaries (11). Both BDNF and NT-4/5 are ligands for the Trk B receptor. Trk B has been demonstrated to be present in mouse oocytes (9) and human fetal oocytes (11). In the present study, we have noted for the first time the presence of NT-4/5 and NT-3 (a Trk C neurotrophin ligand) in the follicular fluid of adult women undergoing IVF.

The presence of Trk B in mouse oocytes (9) and in human fetal oocytes (11), coupled with the finding of NT-4/5 in human follicular fluid and the demonstration that NT-4/5, like BDNF, promotes mouse oocyte meiosis I polar body extrusion, suggests paracrine roles for both BDNF and NT-4/5 within the human follicle. The presence of Trk C in human granulosa cells (12) and the presence of NT-3 in human follicular fluid, along with the absence of Trk C in mouse oocytes, suggest the possibility of an autocrine role for NT-3 in the human ovarian follicle. Together, these data support the concept that the presence of a family of neurotrophins (both Trk B and Trk C ligands) within the human ovarian follicle influence intrafollicular signaling pathways for communication between somatic cells (granulosa-granulosa) as well as between somatic and germ cells (granulosa-oocyte).

In the present study, we did not observe an effect of NT-3 on mouse oocyte GVBD or polar body extrusion, which is consistent with the absence of Trk C receptors in the mouse oocyte. At the same time, NT-4/5, a Trk B ligand, significantly accelerated polar body extrusion relative to control oocytes not exposed to neurotrophin. Although NT-3 has been reported to bind to Trk B receptors (13, 14), we did not observe an effect of NT-3 on polar body extrusion.

At 100 ng/ml, NT-4/5 appears to no longer influence polar body extrusion (as shown in Fig. 1); polar body extrusion was similar to controls at less than 10 ng/ml or 1 ng/ml concentrations. These observations suggest down-regulation of Trk B activity by elevated NT-4/5, as has been reported for BDNF in rat cerebellar cells (15). The low-affinity neurotrophin receptor, p75, also has been reported to inhibit Trk B activity (16), but the mechanisms and physiological significance of these observations remain to be elucidated.

The role(s) of neurotrophins in reproductive tissue remains largely speculative, and information is limited regarding specific neurotrophin effects and mechanisms during gamete development and maturation. However, evidence pointing to the importance of this family of growth factors during gamete development and maturation is rapidly accumulating. For example, in the testis, neurotrophins and their receptors have been clearly associated with spermatogenesis, demonstrating distinct distributions between somatic and germ cells (16, 17). NGF activation of Trk A in cultured thecal cells is reported to be involved in disruption of gap junctions (5) and may stimulate cyclooxygenase II production, progesterone secretion, and cell proliferation (18), events that are associated with ovulation. Whether or not the low-affinity neurotrophin receptor, p75, contributes to regulation oocyte/follicle development and ovulation remains unclear, but p75 appears to modulate Trk activation and may alter binding characteristics of the Trk ligands (19, 20). Our observations suggest that BDNF and/or NT-4/5 and Trk B may influence nuclear maturation of the developing oocyte. Further investigations will refine these relationships and potentially provide new appreciation and understanding of gamete development.

Acknowledgments

Footnotes

This work was supported in part by National Institutes of Health (NIH)–National Institute on Aging Grant AG 15425 (to D.B.S.) and NIH Grant HD 23315 (to C.F.D.).

Abbreviations: BDNF, Brain-derived neurotrophic factor; GVBD, germinal vesicle breakdown; IVF, in vitro fertilization; NGF, nerve growth factor; NT-3, neurotrophin-3; NT-4/5, neurotrophin-4/5.

Received March 29, 2002.

Accepted June 23, 2002.

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