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Department of Obstetrics, Gynecology and Reproductive Sciences (D.B.S., B.F., R.M.S., S.C.), Division of Reproductive Endocrinology and Infertility, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, New Brunswick, New Jersey 08901; and Department of Neuroscience and Cell Biology (C.F.D.), 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.
Abstract
Neurotrophins are a family of soluble polypeptide growth factors widely recognized for their roles in the mammalian nervous system. One such neurotrophin, brain-derived neurotrophic factor (BDNF) was originally described in the nervous system but has now been shown to be expressed in a variety of nonneuronal tissues including endocrine tissues. We examined the human ovarian follicle for its possible secretion of BDNF and further studied mouse oocytes to determine BDNF’s possible influence upon oocyte maturation.
In a series of experiments derived from human specimens from in vitro fertilization following oocyte retrieval, BDNF was detected in human follicular fluid. To define the source of BDNF, cumulus granulosa cells (the cells that immediately surround the developing oocyte) were grown in cell culture for 1–2 d. BDNF protein increased over 24 h in the culture medium. Moreover, the release of BDNF was enhanced upon stimulation with cAMP or forskolin, an activator of cAMP. In contrast, mural granulosa (cells lining the follicle), oocytes, and embryos did not release appreciable quantities of BDNF. To examine possible targets of BDNF, mouse studies were used to localize the BDNF receptor, Trk B, immunocytochemically. The receptor was present on the surface of isolated oocytes. Moreover, BDNF promoted mouse oocyte maturation in culture. These experiments demonstrate for the first time the presence and secretion of BDNF from follicular cells in the human ovary and suggest a possible role for BDNF in the regulation and modulation of oocyte maturation.
NEUROTROPHINS ARE A family of soluble polypeptide growth factors widely recognized for their roles in the mammalian nervous system. They include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-4/5 (NT-4/5), and neurotrophin-3 (NT-3). 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).
Several members of the neurotrophin family (NGF, BDNF, NT-4/5, NT-3) and their respective receptor tyrosine kinases (Trk A for NGF, Trk B for BDNF, and NT-4/5, Trk C for NT-3) have been found to be expressed in the mammalian ovary (3). The actions of neurotrophins within the rodent ovary were initially thought to be limited to their support of ovarian innervation. However, in recent years some of these neurotrophins and their respective receptors have been found to influence rodent ovarian function including ovulation (4, 5), steroid secretion (6), and follicular development (7, 8, 9, 10). Whether these neurotrophins are present and serve similar roles in the human has not been addressed.
Although Trk A and Trk C have been noted in human granulosa cells using immunohistochemistry (11), there has been no report of expression of neurotrophins within the human ovary. We present the first report that a member of the neurotrophin family, BDNF, is localized in the human ovarian follicle. Its specific site of production is identified. Mouse studies suggest a possible function of follicular BDNF.
Materials and Methods
Human studies
Population. Thirty-nine women who were <40 yr of age 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 (12). Transvaginal follicular aspiration was performed under sedation 36 h after hCG injection. Pregnancy was defined as fetal cardiac activity on ultrasound performed 4 wk or more after follicular aspiration. Approval of this study was obtained from the Institutional Review Board at 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 follicular aspiration of 80 follicles from 39 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 BDNF.
Granulosa cell culture study. Additional follicular aspirates were obtained and pooled within a subset of individual patients (n = 18) for the purpose of granulosa cell (mural and cumulus) culture. The follicular fluid was centrifuged 10 min, 700 x g at 20 C. The top layer of cells enriched with mural granulosa was carefully collected into HBSS (Ca and Mg-free). The cell suspension was carefully overlaid on top of 50% Percoll and centrifuged for 20 min at 700 x g. Following Percoll separation, the mural granulosa cell enriched interface layer was collected and washed in HBSS. The resultant mural granulosa cell pellet was incubated in Hanks’ medium containing 2 mM EDTA for 5 min and vortexed at low speed. The mural granulosa cell suspension was further purified with anti-CD45 immunomagnetic beads (Beckman Coulter, Inc., Miami, FL) to remove residual lymphocytes from the cell suspension yielding a greater than 95% pure sample as defined morphologically. Cumulus granulosa cells removed from oocytes in preparation for IVF/intracytoplasmic sperm injection (ICSI) were dissociated in hyaluronidase (100 IU/ml, Sigma, St. Louis, MO) then washed twice with HBSS. Preparation of cumulus cells were of 99% purity.
Both mural (n = 8) and cumulus (n = 18) granulosa cells were plated at 1.5 x 105 cells in 0.3 ml of media in 24-well plates. Mural and cumulus cells were cultured in F-10 medium supplemented with 10% FBS at 37 C, 5% CO2. Culture media was collected at 24 and 48 h and frozen at -80 C for assay of BDNF. For the purpose of examining the regulation of BDNF expression by c-AMP, cumulus cells were combined between two patients for each experiment. After 24 h of initial culture, cells were rinsed three times and changed to fresh media with or without 8-bromo-cAMP (Sigma) at concentrations of 30, 125, and 500 µM, respectively, and forskolin, an activator of cAMP, (10 µM, Sigma). The culture media was collected after 24 h of culture for assay of BDNF.
Human oocyte and embryo culture medium. One hundred forty-seven stripped (without cumulus) oocytes were obtained from 10 patients. Oocytes were pooled within each patient and were incubated 3–5 h at 37 C, 5% CO2 in human tubal fluid (HTF, Conception Technologies, San Diego, CA) microdrops (40 µl/drop) that were supplemented with 10% synthetic serum substitute (Irvine Scientific, Irvine, CA) before ICSI. Following ICSI, oocytes were transferred to culture medium drops under oil, and the HTF drops collected were pooled together to achieve an adequate volume (100 µl) for testing and frozen at -20 C for BDNF assay. Seventy-six zygotes resulted from ICSI and were cultured in Basal XI (Sage Biopharma, Bedminster, NJ) with 10% synthetic serum substitute for 72 h before embryo transfer. One to two zygotes were cultured in each 25-µl drop of culture media. The 72-h embryo culture media were collected and pooled together to achieve an adequate volume (100 µl) for testing and frozen at -20 C for BDNF assay.
BDNF assay. BDNF levels were determined using the commercially available BDNF Emax immunoassay system (Promega Corp., Madison, WI). The ELISAs were performed according to the manufacturer’s protocol. Follicular fluid, granulosa cell culture media oocyte or embryo culture media was added to a 96-well immunoplate precoated with human BDNF specific monoclonal antibody. The plate was incubated at room temperature for 2 h with shaking. Anti-BDNF monoclonal antibody was used as the capture Ab and anti-BDNF pAb was used as reporter Ab. After washing, the amount of specifically bound pAb was detected using a species-specific anti-IgY antibody conjugated to horseradish peroxidase as a tertiary reactant. The 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. The BDNF antibody demonstrated less than 3% cross-reactivity with other related neurotrophic factors (NGF, NT-3, and NT-4) at 100 ng/ml. The detection sensitivity of the ELISA was 15.6 pg/ml with an intrassay coefficient of variation of 2.2% at a mean concentration of 286.1 pg/ml BDNF, according to the manufacturer. Our lab intrassay coefficient variation was 6.3%, and our interassay coefficient of variation for the ELISA assay was 8.6%.
Several negative control experiments were performed to rule out nonspecific binding to human BDNF antibody and potential human IgG from serum contamination that could theoretically cross-react with the ELISA assay. Negative controls included: 1) absence of the reporting antibody specific to BDNF and 2) absence of the reporting antibody and the ligand. These negative controls were included in each assay performed and demonstrated the absence of nonspecific binding.
Mouse studies
Trk B receptor identification in mouse oocytes. Five mice (C57BL/6 mice, Taconic Labs, Germantown, NY), 4–5 wk old, were injected ip with 6–8 IU PMSG (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. Isolated oocytes were transferred into ELISA wells (Removawell, Dynex Technologies, Inc., Chantilly, VA) or 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 paraformaldehyde fixation, oocytes were washed three times with cold PBS and stored in PBS at 4-8 C until stained.
For Trk B receptor staining, PBS was replaced with 15% goat serum in 0.3% Triton-X buffer for 30–60 min at room temperature. Oocytes were then washed three times with PBS at room temperature, followed by overnight incubation at 4-8 C in 1:200 rabbit anti-Trk B antibody that recognizes an intracellular domain of intact Trk B receptor (a gift of Dr. Stuart Feinstein). Following incubation with Trk B antibody oocytes were rinsed three times with PBS then incubated 1 h at room temperature in goat antirabbit IgG (1:200). Oocytes were again rinsed three times with PBS and incubated 1 h in avidin-biotinylated enzyme complex (ABC reagent, Vectastain Elite, Vector Laboratories, Inc., Burlingame, CA) followed by three rinses with Tris buffer. The final Tris buffer was replaced with diaminobenzidine tetrahydrochloride under stereoscopic visualization, recording the time required for oocyte staining to occur. Control oocytes were treated as above except that rabbit anti-Trk B antiserum was omitted.
Effect of BDNF on in vitro maturation of mouse oocytes. Six 4- to 5-wk-old female C57BL/6 mice (Taconic Labs) were given a single ip injection of 7.5 IU PMSG (Sigma). Eighty immature oocytes were harvested 48 h after gonadotropin injection. Cumulus cells were removed mechanically (27-gauge insulin needles) at room temperature. Oocytes were equally divided into control and experimental groups. Immature oocytes (n = 40) were incubated (5% CO2 in air, 37 C) in HTF with 10 ng/ml BDNF (PeproTech, Inc., Rocky Hill, NJ). Control oocytes (n = 40) were cultured in HTF under the same conditions without addition of BDNF. The percentage of oocytes in a cohort of oocytes that demonstrated germinal vesicle breakdown and/or first polar body extrusion was recorded after 6, 20, 24, and 48 h in culture.
Statistical analysis
Statistical analysis of data were performed using an unequal variance two-tail t test, Fisher’s exact test, or 
2 for group comparisons where appropriate. Statistical significance was assumed at P < 0.05.
Results
Human studies
Follicular fluid study. Previous studies suggest that neurotrophins influence ovarian function in rodent models. To begin to define the role of BDNF in the human ovary, initial studies examined follicular aspirates from each of the 39 women. BDNF was demonstrated in all aspirated follicles. The mean follicular fluid concentration of BDNF was 645.2 ± 23.6 pg/ml. After patients were categorized into one of two groups, pregnant (n = 23) and nonpregnant (n = 16). Comparisons were examined for mean age, serum d 3 FSH, serum E2 max, number of retrieved oocytes per patient, number of embryos available for transfer (an indication of the quality of the embryos) and follicular fluid concentrations of BDNF. There were no differences in mean age, serum d 3 FSH, serum E2 max, number of retrieved oocytes per patient, clinical pregnancy rates, or follicular BDNF concentration. However, it is noted that a comparison of clinical pregnancy rates was based on a relatively small sample size and has limited statistical power.
Identification of cells expressing BDNF: granulosa cells (mural and cumulus). BDNF was not detectable in the culture media of mural granulosa in 7 of 8 cell cultures at 24 h and 6 of 8 cultures at 48 h regardless of eventual pregnancy status. One of eight mural granulosa cell cultures had low concentrations of BDNF 70.0 ± 1 pg/ml at 24 h and two of eight had a mean of BDNF 130.5 ± 52.8 pg/ml at 48 h.
In contrast to mural granulosa, BDNF was noted to be present in all (n = 18) samples of cumulus granulosa cell in culture at 24 h with a mean concentration of 268.7 ± 43.5 pg/ml. As shown in Fig. 1
, a time-dependent secretion of BDNF by cumulus cells (n = 18) is suggested by a 56% increase in media BDNF concentration after 48 h (419.50 ± 47.5 pg/ml) compared with 24 h culture (P = 0.03).
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). This observation was confirmed by forskolin, an activator of intracellular adenylate cyclase that elevate intracellular cAMP, which increased BDNF production by 11-fold (P = 0.016) (Fig. 2B).
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Mouse studies
To explore the significance of BDNF in follicular fluid, subsequent studies used a mouse model. Expression of the BDNF receptor, Trk B, and effects of BDNF on oocytes were investigated.
Trk B receptor identification in mouse oocytes. Seventy-five oocytes (43 exposed to anti-Trk B antibody, 32 oocytes exposed to control serum) from five mice were evaluated in three experiments. Three types of stain reaction in the 43 exposed to anti-Trk B antibody were observed: 1) intense, almost black stain within 10–20 sec of diaminobenzidine tetrahydrochloride addition (positive reaction); 2) light pink color appearing over the course of 3–5 min (equivocal staining), suggesting that not all oocytes express Trk B); or 3) no discernible color change after greater than 5 min (negative reaction). Thirty-six oocytes (84%) exposed to anti-Trk B antibody demonstrated intense, positive staining, whereas seven oocytes (16%) demonstrated equivocal or negative staining. None of the control oocytes, incubated without primary antibody, exhibited a positive staining reaction. Twenty-two percent of the control oocytes exhibited equivocal staining whereas 78% of controls demonstrated no color change. The proportion of oocytes stained was significantly greater in the group exposed to anti-Trk B than in controls not exposed to anti-TrkB antibody (P < 0.0001).
Effect of BDNF upon in vitro maturation of mouse oocytes.
There was no difference in the proportion of oocytes demonstrating germinal vesicle body breakdown in the presence of, or absence of, BDNF at any time point. However, the percentage of oocytes demonstrating first polar body extrusion was higher after 6 h in the presence of 10 ng/ml BDNF than in the control oocytes. Significantly higher rates of PB extrusion in the presence of BDNF were seen at 20 h (2.5% vs. 17.5%, P = 0.025), 24 h (5% vs. 20%, P = 0.043), and 48 h (5% vs. 20%, P = 0.043) for control and treated oocytes, respectively (Fig. 3).
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Our interest in exploring the role of neurotrophins in the human ovary was initiated when we considered the physiologic similarity between the basic structural unit of the brain (neuron/glia) and the basic structural unit of the ovary (follicle containing oocyte/granulosa). In the brain, glia serve as the supporting tissue and participate in the two-way communication via neurotrophins with its respective functional unit, the neuron (13). Our data support the possibility that a similar dynamic is present within the ovary. The supporting tissue (granulosa) within the follicle participates in the two-way communication via growth factors with its respective symbiotic unit, the oocyte. In the brain, estrogen has been shown to affect the expression of BDNF mRNA during neural development and aging (14, 15, 16). A similar dynamic may occur within the follicle between estrogen production by granulosa and its influence upon oocyte development and maturity. Given these functional similarities as well as the evidence for a role of neurotrophins within human nonneural tissues and its previously described roles in the rodent ovary, we examined whether or not a neurotrophin, specifically BDNF, is present within the human ovary.
Before this report, no member of the neurotrophin family had been reported to be present within the human ovary. Our data demonstrate for the first time the presence of BDNF in the human ovarian follicle. More specifically, BDNF was noted to be present within the follicular fluid of preovulatory follicles of women undergoing IVF. The site of BDNF secretion appears to be the cumulus granulosa as illustrated by rising concentrations of BDNF in cumulus culture at 48 h compared with concentrations at 24 h. This is further supported by evidence that a cAMP analog stimulates the secretion of BDNF by cumulus cells in a dose-response fashion. Similarly, the cAMP activator, forskolin, also resulted in increased secretion of BDNF by cumulus cells. Mural granulosa, oocytes, and embryos do not appear to secrete BDNF reliably in measurable amounts.
Due to constraints of the use of human oocytes, we considered the mouse model to elucidate the possible relevance of BDNF to oocyte development. Specifically, we were able to demonstrate the presence of Trk B in the gonadotropin stimulated mouse oocyte. Further studies will be necessary to determine the natural history of the BDNF receptor in oocytes and the nature of postreceptor signaling pathways. Additional studies will also be necessary to demonstrate Trk B receptor in human oocytes. Data from these mouse experiments suggest that BDNF may be important for oocyte maturation, but the mechanisms underlying this role have not yet been elucidated.
Differentiation and survival of neurons has been shown to be influenced by a host of growth factors including BDNF, NGF, ciliary neurotrophic factors, and fibroblast growth factors. While little is currently known about the role(s) of BDNF in the cumulus-oocyte complex, there are a number of possibilities based on neurotrophic factor activity in other cell systems. For example, neural crest cells respond to NGF by halting cell cycle functions; the resulting cells then undergo a differentiation process involving the extension of neurites (17). On the other hand, in oligodendrocytes of the central nervous system neurotrophins have been reported to enhance DNA synthesis (18, 19). In the nervous system, neurotrophins also have been reported to enhance survival or cell death (20, 21) under different circumstances.
Our data support the possibility that BDNF enhances the extrusion of polar bodies, suggesting that it may increase the rate of meiosis. It may be speculated that neurotrophic factors may also be involved in the differentiation of primitive granulosa cells into specialized cumulus cells with cellular process extending through the zona pellucida to the oolemma. Neurotrophic factors may also be involved in oocyte/follicle selection mechanisms. Hence, it is speculated that neurotrophic stimulation of specific oocyte release may be involved in selective atresia of the developing follicle cohort.
Identification of Trk B receptor in the unfertilized, preovulatory mouse oocyte suggests that neurotrophic factors, specifically BDNF, may have a paracrine function in promoting granulosa-oocyte communication within the mammalian follicle. Based upon the results of these experiments, we propose a hypothetical model of the gonadotropin-granulosa cell-oocyte interaction involving BDNF. Endogenous serum gonadotropins bind to cumulus granulosa cells resulting in activation of cAMP, which leads to production and secretion of BDNF within the cell. The cumulus produced BDNF is secreted into the follicular fluid and binds with the Trk B receptor located on the surface of the oocyte. Once binding of the Trk B oocytes and its ligand takes place, altered cell cycle kinetics occur eventually resulting in enhanced maturation of the oocyte.
Recent studies using knockout mice by Ojeda et al. (10) and Dissen et al. (22) support the role BDNF and additional neurotrophins (i.e. NT-4, NGF) may play in somatic-germ cell communication in the organization of oocyte and pregranulosa into forming primordial follicles. Thus, our human and mouse data may represent a continuum of the paracrine role of BDNF in the somatic-germ cell communication which occurs in both the initial basic organization of the primordial follicle as well as in the process of folliculogenesis in preparing a fertilizable oocyte. Additional identification of Trk B within cumulus granulosa would support an autocrine role for BDNF within the human ovary. Further study of human ovarian physiology may reveal a significant role for ovarian neurotrophins in the regulation and modulation of the female reproductive system.
Acknowledgments
We gratefully acknowledge the assistance of Denise Smith and Lauren Lercher.
Footnotes
This work was supported in part by NIH-NIA Grant AG-15425 (to D.B.S.) and NIH HD-23315 (to C.F.D.).
Abbreviations: BDNF, Brain-derived neurotrophic factor; HTF, human tubal fluid; ICSI, intracytoplasmic sperm injection; IVF, in vitro fertilization; NGF, nerve growth factor; NT-4/5, neurotrophin-4/5; NT-3, and neurotrophin-3.
Received August 15, 2001.
Accepted October 26, 2001.
References
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