DNA Microarray Analyses. Mice (n = 108) were injected at 21 days of age with Humegon (7.5 units per animal, Organon) containing follicle-stimulating hormone and LH activities to stimulate follicular growth. Forty-eight hours later, some animals were treated i.p. with Pregnyl (5 units per animal) containing LH activity to induce ovulation. Ovaries were dissected from animals killed bi-hourly after Humegon treatment (three mice per group) and hourly after Pregnyl treatment (one mouse per group) for RNA extraction (TRIzol, Invitrogen). Aliquots of 6 μg of total RNA at 1 μg/μl for one-chipset hybridization were stored at -80°C. Samples were hybridized to the Affymetrix mouse MGU74v2 arrays A, B, and C according to standard Affymetrix protocols. The pooled follicular phase samples were hybridized in duplicate, and the postPregnyl samples were single determinations.

Follicle Cultures. Preovulatory follicles were excised from mouse ovaries 48 h after PMSG treatment and cultured to examine nuclear maturation of oocytes (2). Follicles (20-30 per vial) were cultured with or without recombinant human BDNF (Pepro-Tech, Rocky Hill, NJ) or 5 μg/ml LH (Organon) in Leibovitz's L-15 medium (Invitrogen). The vials were flushed at the start of the culture with O₂/N₂ (at a 1:1 ratio), sealed, and cultured at 37°C with gentle shaking for 6 h. After culture, cumulus oocyte complexes (COCs) were isolated, and, after cumulus cells were removed, oocytes were examined for the occurrence of GVBD.

Evaluation of First Polar Body Extrusion. For evaluating the transition from metaphase I stage to metaphase II (MII) stage oocytes, COCs were obtained from mouse ovaries 48 h after PMSG treatment by puncturing the largest follicles in M2 medium (Specialty Media, Phillipsburg, NJ). COCs were washed twice, transferred to modified M16 medium (Specialty Media) without FBS, and cultured with or without different doses of BDNF, mouse nerve growth factor (NGF; R & D Systems), or human neurotrophin-3 (NT-3) (R & D Systems) for 20 h at 37°C in 5% CO₂/95% air. Some COCs were also cultured with BDNF with or without the TrkB ectodomain (R & D Systems), pan-specific Trk receptor inhibitor K252a (Calbiochem) (10), or K252b (11). The occurrence of first polar body extrusion in the oocyte was examined after removing cumulus cells by using a small-bore pipette under Hoffman contrast microscopy (Nikon).

In Vitro Maturation, Fertilization, and Early Embryonic Development. We performed in vitro maturation of oocytes followed by in vitro fertilization as described in ref. 12 with slight modifications. COCs from PMSG-primed mice were obtained in the M2 medium supplemented with 5% FBS before culturing in minimum essential media-α (Invitrogen) supplemented with Earle's salts, 10 μg/ml streptomycin sulfate, 75 μg/ml penicillin G, and 5% FBS in the presence or absence of 3 ng/ml BDNF at 37°C in 5% CO₂/95% air. After 16 h of treatment, cumulus cells were removed and the oocytes were examined and classified according to their developmental stage (germinal vesicle, metaphase I, or MII). MII-stage oocytes were inseminated with sperm from B6D2F1 males and incubated for 4 h at 37°C in 5% CO₂/95% air. After in vitro fertilization, fertilized oocytes were recovered, washed three times, and cultured in human tubal fluid medium (Specialty Media). The following morning, two-cell stage embryos were collected and cultured in 2 ml of modified M16 medium for 5 days more up to the blastocyst stage at 37°C in 5% CO₂/95% air. Embryonic development was monitored daily by Hoffman modulation contrast microscopy, and the progression of fertilized eggs to preimplantation embryos was assessed.

In Vivo Analysis. To evaluate first polar body extrusion, immature mice were treated with 7.5 units of PMSG followed by 10 units of hCG 48 h later with or without an i.p. injection of K252a (dissolved in 10% dimethyl sulfoxide/0.9% saline). For negative controls, the plasma membrane nonsoluble K252b was used. Twelve hours after hCG injection, ovulated COCs were obtained from the oviducts. After treatment with hyaluronidase (Specialty Media) for 1-2 min, oocytes were separated from cumulus cells and the proportion of oocytes showing first polar body extrusion was evaluated. To evaluate the role of BDNF in the conditioning of oocytes for early embryo development, immature mice were treated with PMSG, followed by hCG as described above. Immediately after hCG injection, animals were allowed to mate before collection of fertilized oocytes 22 h later. To ensure the suppression of Trk receptor activity, 10 μg of K252a or K252b was administrated i.p. at 0, 4, and 8 h after hCG injection. Fertilized oocytes were separated from cumulus cells, washed three times in M2 medium, and cultured for 5 days in modified M16 medium. Embryonic development was monitored daily, and the progression of fertilized eggs into preimplantation embryos was assessed.

By using isolated ovarian cells, RT-PCR was performed to examine cells expressing BDNF and TrkB in the mouse ovary. We also examined the expression of the low-affinity receptor p75NTR for neurotrophins. Expression of the BDNF transcript was detected in mural granulosa and cumulus cells but not in oocytes from mice primed with PMSG for 48 h (Fig. 2A). In contrast, the TrkB mRNA was expressed exclusively in oocytes, although the p75NTR transcript was found in granulosa cells, oocytes (Fig. 2 A), and the theca shell (data not shown). We further confirmed the localization of BDNF and TrkB proteins by using immunohistochemistry. As shown in Fig. 2B and Fig. 5, strong BDNF staining was observed in mural and cumulus granulosa cells of preovulatory follicles at 7 h after hCG treatment, and a weaker signal was found in mural granulosa cells in small antral follicles. In addition, TrkB expression in the plasma membrane of the oocyte was confirmed by using immunofluorescence staining (Fig. 2C).

Based on hCG stimulation of BDNF expression in ovarian somatic cells and the exclusive expression of TrkB in oocytes, we hypothesized that BDNF acts as a paracrine factor to regulate oocyte function. In cultured preovulatory follicles, treatment with LH, but not BDNF, for 6 h induced GVBD in oocytes (Fig. 3A). Furthermore, treatment with BDNF, unlike LH, did not induce cumulus cell expansion (data not shown). Because oocytes obtained from preovulatory follicles underwent spontaneous GVBD when cultured as COC, we used the COC model to test the effect of BDNF to promote further oocyte development. As shown in Fig. 3B, treatment with BDNF, but not the related NGF or NT-3, increased first polar body extrusion in cultured oocytes in a dose-dependent manner. In addition, the stimulatory effect of BDNF was blocked by cotreatment with the TrkB ectodomain (Fig. 3C), whereas treatment with the TrkB ectodomain alone was ineffective. We further used the Trk receptor inhibitor K252a to block TrkB function in the oocyte. As shown in Fig. 3C, concomitant treatment with K252a, but not the membrane nonsoluble K252b, suppressed the stimulatory effect of BDNF on first polar body extrusion. Because BDNF, but not related neurotrophins, are increased during the preovulatory period (data not shown), we further used K252a to examine the role of endogenous BDNF on oocyte function in vivo. Although treatment with K252a or K252b did not affect the number of ovulated oocytes per animal (vehicle, 40.2 ± 7.5; K252a-treated, 37.8 ± 5.3; K252b-treated, 38.4 ± 8.7), treatment with K252a inhibited by 30% the first polar body extrusion by ovulated oocytes (Fig. 3D). In contrast, similar treatment with K252b was ineffective. Of interest, >95% of ovulated oocytes from all groups showed GVBD accompanied by expansion of the cumulus cells.

The role of BDNF in conditioning the oocytes for subsequent fertilization and progression to blastocysts was evaluated in vitro (Fig. 4). We cultured COCs obtained from mice primed for 48 h with PMSG and treated them with or without BDNF. To avoid hardening of the zona pellucida that is unfavorable for in vitro fertilization, 5% FBS was included for all cultures. Similar to serum-free cultures (Fig. 3B), treatment with BDNF increased more than 2-fold the proportion of oocytes showing first polar body extrusion (Fig. 4A, MII-stage oocytes). These MII oocytes were then fertilized in vitro without further treatment with BDNF. As shown in Fig. 4A, pretreatment with BDNF increased by 2- and 5-fold the proportions of MII oocytes that developed into the two-cell and blastocyst-stage embryos, respectively. Because the oocyte content of glutathione is important to sperm nuclear decondensation (13), the glutathione concentration in oocytes also was evaluated. As shown in Fig. 4B, glutathione levels were low in germinal vesicle-stage oocytes and in oocytes that spontaneously completed first polar body extrusion in vitro. However, treatment with BDNF increased glutathione content in MII oocytes (Fig. 4B).

Four of the five known neurotrophins, including NGF, BDNF, NT-3, and NT-4/5, and their receptors (TrkA, TrkB, Trk C, and p75 NFR) are expressed in early ovarian follicles (14). Mice defective in the expression of TrkB or its ligands (BDNF and NT-4/5) exhibited arrest in follicle development at the primary follicle stage (8, 9). Furthermore, treatment with the Trk receptor inhibitor, K252a, or the combined addition of antibodies against BDNF and NT-4/5 decreased primordial follicle survival in vitro (9). Although the expression of TrkA and its ligand, NGF, are increased in the thecal cells of preovulatory follicles and immunoneutralization of NGF actions inhibit follicle rupture (15, 16), the role of BDNF and its receptor, TrkB, in preovulatory follicles is unknown.

DNA microarray analyses of the ovarian transcriptome during the preovulatory period allowed us to identify major stimulation of BDNF expression after the LH/hCG induction of ovulation. Our findings of gonadotropin stimulation of ovarian BDNF secretion in mice in vivo are consistent with an earlier study using cultured human cumulus cells (17). BDNF acts on the TrkB receptors. Although kinase domain-containing TrkB receptors are expressed only at low levels in the oocytes and granulosa cells of primordial and growing follicles in mice (8), our data demonstrating TrkB expression in oocytes of preovulatory mouse follicles are consistent with an earlier work with humans (18). Ovarian localization of these genes indicated that BDNF produced by mural and cumulus granulosa cells is a paracrine factor acting on the TrkB receptor that is expressed exclusively in the oocyte.

Completion of nuclear maturation involves GVBD and extrusion of the first polar body. Although treatment of cultured COCs with BDNF did not affect GVBD, it facilitated first polar body extrusion, consistent with an earlier finding (18). We further demonstrate that the stimulatory effect of BDNF was blocked by the soluble ectodomain of TrkB or the Trk receptor inhibitor, K252a, suggesting mediation by the TrkB receptor. The important role of endogenous BDNF in promoting first polar body extrusion is underscored by the suppressive effects of the Trk receptor inhibitor, but not the related K252b, in ovulating animals in vivo. Because treatment with the Trk inhibitor did not affect GVBD of ovulated oocytes in these animals, it is apparent that sequential steps of nuclear maturation of the oocyte are controlled by different paracrine factors. In addition to improving the in vitro maturation of oocytes for successful in vitro fertilization, elucidation of the molecular mechanisms underlying ovarian BDNF actions could also allow the formulation of novel contraceptive strategies.

Some oocytes competent to complete nuclear maturation are unable to develop into the blastocyst stage, which is indicative of deficient or defective cytoplasmic maturation of the oocyte (3). During maturation of preovulatory oocytes, cytoplasmic changes in oocytes are necessary to allow for the acquisition of the maternal components required for optimal development of fertilized oocytes into preimplantation embryos. We demonstrated that in vitro treatment of COCs with BDNF not only augmented first polar body extrusion but also enhanced the subsequent development of MII oocytes to two-cell and blastocyst embryos. Furthermore, in vivo treatment with the Trk receptor inhibitor indicated the essential role of preovulatory increases of endogenous BDNF in the cytoplasmic maturation of the oocyte, thereby showing a major suppression of embryos capable of developing into the blastocyst stage. Even for embryos developed to the blastocyst stage, pretreatment with the Trk receptor inhibitor decreased their cell numbers by half. Furthermore, oocytes that spontaneously reached the first polar body stage had lower levels of glutathione (Fig. 4B), which is believed to be important in the fertilization of MII oocytes by facilitating sperm nuclear decondensing activity (13). Because BDNF treatment enhanced oocyte glutathione content, these findings suggest that BDNF could play a role in successful fertilization. Our data are consistent with earlier studies showing a correlation between glutathione levels in mature pig oocytes and their ability to form male pronuclei after fertilization (19). Thus, ovarian BDNF could activate separate downstream pathways in the oocyte to facilitate nuclear maturation (first polar body extrusion) as well as fertilization (glutathione content) and early embryo development.

In addition to the promotion of GVBD, treatment with EGF also increases first polar body extrusion (20, 21) and the number of cells in the blastocyst (22). Because the expression of three EGF-like factors, in a manner similar to BDNF, is increased after the preovulatory LH surge (1), these ovarian paracrine factors could act synergistically to promote first polar body extrusion. In addition, meiosis-activating sterol, activin, and a heparin-binding growth factor, midkine, also were found to promote the developmental competence of cultured mouse and bovine oocytes (12, 23, 24). Although the physiological significance of these three factors during the preovulatory period is unclear, it is becoming apparent that redundant intraovarian pathways in oocytes (TrkB and LGR8) and cumulus cells (EGF receptors) are activated during the preovulatory period to ensure successful oocyte maturation, fertilization, and early embryo development. Gene expression during oocyte maturation, fertilization, and early embryo development is regulated mainly by translational activation of maternally derived mRNAs, and the proper conditioning of the oocyte cytoplasm enables the development of totipotent blastocysts. Future studies on the translation of oocyte mRNAs regulated by BDNF and other oocyte-conditioning factors could provide clues regarding the molecular mechanisms underlying the unique developmental potential of fertilized oocytes. These studies could also shed light on the capacity of the oocyte cytoplasm to reprogram substituted nuclei from somatic cells after nuclear replacement or cloning (25, 26).