{"paper_id":"ecfddbaf-5e24-49ce-aac4-8d61e50f67a1","body_text":"The mammalian ovary is a highly innervated organ that contains sympathetic and sensory nervous system fibers. These nerve fibers can reach most of the intraovarian compartments, including the ovarian artery, interstitial tissue and ovarian follicle ( Malamed  et al. , 1992 ). A rat ovary that was transplanted to an ectopic site was promptly reinnervated by both sympathetic and sensory nerve fibers ( Lara  et al. , 1991 ), indicating that the ovary could produce certain substances that promote the rapid ingrowth of such nerve fibers from the surrounding tissues. Indeed, evidence supported by animal studies showed that the innervated rat ovary was able to produce and secrete nerve growth factor (NGF), which was stimulated and regulated by the innervation ( Lara  et al. , 1990 ). In the rhesus monkey ovary, a two-fold increase in the density of ovarian innervation occurred during prepubertal development, suggesting that NGF exerts its trophic effects on the development of ovarian sympathetic neurons that contribute to ovarian development and reproductive capacity ( Schultea  et al. , 1992 ). During mammalian development, the tissue-derived neurotrophic and neural guidance factors are essential to determine the proper directionality of the related tissues and organs, including heart, intestine and airways ( Keast, 2013 ). Dysregulation of these neurotrophic and neural systems can cause defects of innervation that have been associated with various human neurological diseases, including Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, neuroblastoma, multiple sclerosis-like model of experimental allergic encephalomyelitis, peripheral neuropathies and even motion sickness ( Calissano  et al. , 2010 ).\nNeurotrophic factors are divided into the following main families: neurotrophins (NTs) and glial cell line-derived neurotrophic factor (GDNF;  Airaksinen and Saarma, 2002 ;  Chao, 2003 ). NTs were initially discovered as molecules that can promote the survival of developing neurons that newly innervate or terminate in their target peripheral tissues; these molecules constitute a class of polypeptide growth factors that are critical for the development, maintenance and regeneration of the central and peripheral nervous system ( Hamburger, 1952 ). Since then, neurotrophic factors and their putative receptors have also been found to play substantial roles in various peripheral tissues, including the highly innervated ovary. Furthermore, experimental data gathered from clinical samples have demonstrated that these neurotrophic factors, including brain-derived neurotrophic factor (BDNF) and GDNF, are highly expressed in the human granulosa cells (GCs) in the absence of innervation ( Zhao  et al. , 2011 ). Similar to the bidirectional communication between neurons and glial cells, NTs, together with other hormones and growth factors, exert a considerable effect on both germ cells and somatic cells during the developmental stages of folliculogenesis ( Davies and Wright, 1995 ;  Dissen  et al. , 2009 ). Over the past few decades, the effects of NTs on growing ovarian follicles have been well studied with regard to follicular assembly, growth, survival and apoptosis (for reviews, see  Ojeda  et al. , 2000 ;  Dissen  et al. , 2002 ,  2009 ). Furthermore, the involvement of NTs and GDNF has been demonstrated in the regulation of the final stage of folliculogenesis, which is characterized by oocyte maturation, steroidogenesis, ovulation and corpus luteum formation; the end point of this process is the acquisition of oocyte competence for embryo development following fertilization (for reviews, see  Linher-Melville and Li, 2013 ). However, the functional roles of NTs and GDNF in the regulation of ovarian function remain poorly defined in humans because most information has been obtained from animal models, even though the fundamental process of ovarian folliculogenesis and oocyte maturation is highly similar across mammalian species. Therefore, in this review, we have summarized the functional roles of NTs and GDNF in human ovarian biology and ovarian disorders. Furthermore, we described and proposed the development of novel strategies for diagnosing, treating and preventing related abnormalities.\n\nComprehensive literature searches using either PubMed, MEDLINE or Google Scholar were performed to review articles written in English focusing on the role of NTs and GDNF in follicle assembly, primordial follicle activation, follicular growth and development, oocyte maturation, steroidogenesis, ovulation and corpus luteum formation, as well as ovarian disorders. The search included all relevant mammals, especially human studies published before August 2018.\n\nThe NTs are a family of soluble polypeptide growth factors that comprises five members, including NGF, BDNF, neurotrophin-3 (NT-3), neurotrophin-4 (NT-4, also known as NT-4/5 or NT-5) and neurotrophin 6 (NT-6), which share approximately 50% amino acid sequence identity ( Skaper, 2018 ). To date, NT-6 has been identified only in non-mammalian species, such as fish ( Götz  et al. , 1994 ). Initially synthesized as pre-pro-proteins in the endoplasmic reticulum, NT precursors are cleaved off the signal peptide and converted into pro-NTs. Prior to their release from the cell, pro-NTs are proteolytically processed by proprotein convertase subtilisin/kexin (PCSK) enzymes that dimerize to their mature forms in the  trans -Golgi network ( Lessmann and Brigadski, 2009 ). In certain tissues, the extracellular pro-BDNF can be cleaved by plasmin, which is activated by tissue plasminogen activator ( Pang  et al. , 2004 ). Mature NTs are non-covalently linked homodimers that are formed by two identical peptide chains of approximately 120 amino acids. Each monomer contains six highly conserved cysteine residues that form a cysteine knot and stabilize the conformation of the proteins ( McDonald  et al. , 1991 ).\nBoth pro- and mature NTs initiate their biological actions by binding to the following principal receptor types: high-affinity transmembrane tropomyosin receptor kinase (Trk) receptors and a low-affinity p75 neurotrophin receptor (p75NTR). High-affinity Trk receptors are encoded by members of the Trk proto-oncogene family that exhibit ligand-dependent activation of tyrosine kinase receptor activity, thus functioning as signaling receptors ( Barbacid, 1994 ;  Longo and Massa, 2013 ). Trk receptors contain a tyrosine kinase intracellular domain and extracellular domain (with a leucine- and cysteine-rich motif) that confers ligand binding ( Yano and Chao, 2000 ). The intracellular action specificity of NTs is determined by the selective interaction with their cognate Trk receptors, TrkA (also known as NTRK1) for NGF, TrkB (also known as NTRK2) for BDNF and NT-4, and TrkC (also known as NTRK3) for NT-3 ( Yancopoulos  et al. , 1990 ;  Barbacid  et al. , 1991 ;  Raffioni  et al. , 1993 ;  Chao, 2003 ) (Table  I ). Notably, NT-3 can also bind to non-cognate Trk receptors (TrkA or TrkB), although the physiological role of this ligand-receptor interaction is not clear ( Benedetti  et al. , 1993 ) (Table  I ).\n Table I Ligand preferences of neurotrophin/glial cell line-derived neurotrophic factor receptors. Receptor Ligand References TrkA NGF, NT-3 a Yancopoulos  et al.  (1990) ,  Benedetti  et al.  (1993) TrkB BDNF, NT-4, NT-3 a Yancopoulos  et al.  (1990) ,  Benedetti  et al.  (1993) TrkC NT-3 Yancopoulos  et al.  (1990) p75NTR NGF, BDNF, NT-3, NT-4 Chao (2003) GFRα1 GDNF Takahashi (2001) RET GDNF, NRTN, ARTN, PSPN Takahashi (2001) ,  Airaksinen  et al.  (1999) a Ligand has lower affinity or is less commonly important for receptor activation  in vivo . Trk, transmembrane tropomyosin kinase; NGF, nerve growth factor; NT-3, neurotrophin-3, BDNF, brain-derived neurotrophic factor; NT-4, neurotrophin-4; p75NTR, p75 neurotrophin receptor; GDNF, glial cell line-derived neurotrophic factor; GFRα1, GDNF receptor-α1; NRTN, neurturin; ARTN, artemin; PSPN, persephin.\nLigand preferences of neurotrophin/glial cell line-derived neurotrophic factor receptors.\na Ligand has lower affinity or is less commonly important for receptor activation  in vivo .\nTrk, transmembrane tropomyosin kinase; NGF, nerve growth factor; NT-3, neurotrophin-3, BDNF, brain-derived neurotrophic factor; NT-4, neurotrophin-4; p75NTR, p75 neurotrophin receptor; GDNF, glial cell line-derived neurotrophic factor; GFRα1, GDNF receptor-α1; NRTN, neurturin; ARTN, artemin; PSPN, persephin.\nIn contrast to the Trk family of receptors, the low-affinity NT receptor p75NTR (also known as the pan-neurotrophin receptor) binds all NTs with equal affinity ( Chao, 2003 ;  Kashyap  et al. , 2018 ) (Table  I ). The p75NTR is a type I transmembrane glycoprotein receptor that is structurally related to the tumor necrosis factor (TNF) receptor family ( Chao, 2003 ). Unlike Trk receptors, p75NTR lacks intrinsic enzyme activity but instead recruits signal adaptors that modulate Trk signaling ( Longo and Massa, 2013 ). Ligand preferences of NT receptors are listed in Table  I .\n\nFirst identified as a trophic factor that promotes the survival of midbrain dopaminergic neurons during embryonic development, GDNF is another neurotrophic factor that has been shown to play an essential role in the development and maintenance of the central and peripheral nervous system ( Lin  et al. , 1993 ;  Tomac  et al. , 1995 ). As distant relatives of the transforming growth factor β (TGF-β) superfamily, the GDNF family comprises four structurally related members, including GDNF, neurturin, artemin and persephin ( Airaksinen and Saarma, 2002 ). Despite low amino acid sequence identity (<20%), GDNF and members of the TGF-β superfamily have similar structural conformations that belong to the cysteine-knot protein family and function as homodimers ( Ibáñez, 1998 ). Similar to NTs, GDNF is initially produced in the form of a precursor, pre-pro-GDNF, in which the signal sequence is cleaved on secretion, and activation of pro-GDNF most likely occurs through proteolytic cleavage by PCSK enzymes (specifically PCSK3, also known as furin;  Airaksinen and Saarma, 2002 ). Animal studies have shown that GDNF can bind heparan sulfate proteoglycans of the extracellular matrix, which increases its local concentration by restricting diffusion in the tissue ( Hamilton  et al. , 2001 ). The human GDNF gene contains six exons and generates two conserved isoforms by alternative splicing of exon 3, including a full-length pre-α-pro-GDNF (α-GDNF) and the shorter pre-β-pro-GDNF (β-GDNF) ( Grimm  et al. , 1998 ) (Fig.  1 ). Compared to the full-length isoform, the short isoform lacks 26 amino acids in the pro-region ( Airavaara  et al. , 2011 ) (Fig.  1 ). However, the lack of amino acids does not affect the proteolytic cleavage site, with both isoforms cleaved into mature GDNF. Recent studies have shown that both GDNF isoforms have a comparable biological effect and efficacy in the central nervous system ( Penttinen  et al. , 2018 ).\nSchematic representation of the human GDNF gene and two splicing protein isoforms . The human glial cell line-derived neurotrophic factor (GDNF) gene has six exons (color boxes represent exons, although not to scale) that encode the pre- α-pro-GDNF isoform with a full-length 58 amino acid pro-region and the pre-β-pro-GDNF isoform with a shorter 32 amino acid pro-region. Adapted from ( Penttinen  et al. , 2018 ).\nIn vitro  studies using overexpression approaches revealed that both GDNF isoforms are secreted from cells in two different ways. α-GDNF and its corresponding mature GDNF proteins are secreted constitutively, whereas the secretion of the β-GDNF (and its related mature GDNF) proteins appears to be activity-dependent ( Lonka-Nevalaita  et al. , 2010 ). Despite the different secretory patterns, the two major isoforms of GDNF are usually expressed in the same tissues but in varying proportions ( Suter-Crazzolara and Unsicker, 1994 ). In the mammalian ovary, the LH surge dramatically induces the expression and secretion of GDNF, which promotes maturation of the oocyte ( Zhao  et al. , 2011 ). Furthermore, the treatment of human GCs with hCG alone or combined with FSH significantly increased GDNF mRNA levels  in vivo  and  in vitro , indicating the positive regulatory role of LH and hCG in the production of GDNF ( Zhao  et al. , 2011 ). However, our most recent studies have shown a potential physiological function exerted by an intraovarian growth factor; bone morphogenetic protein 2 (BMP2), which negatively regulates the expression of GDNF in human granulosa-lutein cells ( Zhang  et al. , 2018 ).\nGDNF signals through a unique multicomponent receptor complex that consists of the GDNF family receptor-α1 (GFRα1) and RET receptor tyrosine kinase ( Takahashi, 2001 ). GFRα1 receptor is usually linked to the plasma membrane by a glycosyl phosphatidylinositol anchor; however, it can be cleaved by an unknown phospholipase or protease that produces soluble forms of these coreceptors ( Paratcha  et al. , 2001 ). GDNF binds to GFRα1 and then forms a complex with RET as a signaling component (Table  I ). First discovered as a proto-oncogene, RET is a single-pass transmembrane protein that contains two main cellular portions ( Takahashi, 2001 ). The extracellular portion consists of four cadherin-like repeats, a cysteine-rich domain and a calcium-binding site, while the intracellular portion comprises a characteristic structural feature of tyrosine kinase domain ( Airaksinen and Saarma, 2002 ).\n\nUpon NT binding, Trk receptors are tyrosine kinase receptors that subsequently trigger the activation of several intracellular signaling pathways, including Ras/mitogen-activated protein kinase (MAPK), phosphoinositide-specific phospholipase C-γ1 (PLCγ1) and phosphoinositide 3-kinase/protein kinase B-mammalian target of rapamycin (PI3K/Akt-mTOR) pathways ( Chao, 2003 ) (Fig.  2 ). Additionally, all four NTs bind to low-affinity p75NTR, which belongs to a family of transmembrane molecules that include receptors for the TNF family ( Barbacid, 1994 ;  Bibel  et al. , 1999 ) (Fig.  2 ).\nNeurotrophin receptors and their downstream signaling pathways . NGF binds to TrkA, BDNF and NT-4 bind to TrkB, and NT-3 binds to TrkC, which further induces the activation of Ras/MAPK, PLCγ1 and PI3K/Akt pathways. All neurotrophins (NGF, BDNF, NT-3 and NT-4) also bind to p75NTR receptor, which initiates the recruitment of various adaptors that further activate NF-κB and JNK pathways. BDNF, brain-derived neurotrophic factor; JNK, Jun kinase; MAPK, mitogen-activated protein kinase; NGF, nerve growth factor; NT-3, neurotrophin-3; NT-4, neurotrophin-4; NF-κB, nuclear factor-κB; PI3K, Phosphatidylinositol-3-kinase; PLCγ1, phospholipase Cγ1.\nFollowing the binding of NTs to their corresponding Trk receptors, the activation of Ras protein occurs, which is an initial response required for cell survival and differentiation. The signal activation cascade is mediated by the binding of an adaptor molecule, Shc, to the phosphorylated tyrosine 490 where the phosphorylated Shc recruits an adaptor protein growth factor receptor-bound protein 2 (Grb2) and a guanine nucleotide exchange factor son of sevenless (SOS;  Nimnual  et al. , 1998 ;  Robinson  et al. , 2005 ). The Grb2-SOS complex further increases Ras-GTP levels by catalyzing nucleotide exchange on Ras ( York  et al. , 2000 ). This activated Ras molecule can stimulate a signaling cascade through various cytoplasmic kinase proteins, including a kinase specific for serine/threonine (Raf), MAPK kinase activator, MAPK and extracellular signal-regulated kinase 1/2 (ERK1/2;  Wood  et al. , 1992 ;  Xing  et al. , 1998 ). The activation of ERK1/2, which further phosphorylates and activates a variety of kinases and nuclear transcription factors, is critical for many Ras-induced cellular responses ( Liebmann, 2001 ). Indeed, the Ras/MAPK pathway mediates various NT-induced regulations of ovarian function in humans ( Dissen  et al. , 2009 ;  Julio-Pieper  et al. , 2009 ).\nIn addition to the Ras/MAPK pathway, one of the main mechanisms that mediates NT/Trk receptor interactions is driven by PLCγ1, which is an intracellular messenger that transmits membrane receptor signaling to several molecules ( Reichardt, 2006 ). The activation of PLCγ1 leads to cleavage of the membrane lipid phosphatidylinositol 4,5-bisphosphate to form inositol trisphosphate (IP3) and diacylglycerol (DAG), two key secondary messengers used in signal transduction in most cells ( Reichardt, 2006 ). IP3 is a soluble molecule and is capable of diffusing to the rough endoplasmic reticulum where IP3 can bind to its receptor and trigger the release of Ca 2+  into the cytoplasm ( Taylor and Tovey, 2010 ). The increase in intracellular Ca 2+  or DAG concentration may stimulate the activity of protein kinase C, which plays essential roles in several signal transduction cascades ( Wilson  et al. , 2015 ;  Ali  et al. , 2016 ).\nThe PI3K/Akt-mTOR signaling pathway integrates both extracellular and intracellular signals that serve as a principal regulator of various cell functions, including cell growth, proliferation, metabolism and survival ( Laplante and Sabatini, 2009 ). In the mammalian ovary, this signaling pathway is the most studied pathway that governs the activation of primordial follicles ( Adhikari and Liu, 2009 ). Recent studies have provided evidence showing that the PI3K/Akt-mTOR signaling pathway and KIT ligand play indispensable roles in the co-ordination of GCs and oocytes, which initiate the activation of primordial follicles in adult ovaries ( Zhang and Liu, 2015 ). Notably, the achievement of this regulation over the reproductive lifespan involves several extracellular intraovarian factors, including NTs, KIT ligand, BMP4, BMP7, vascular endothelial growth factor (VEGF) and other molecules ( Adhikari and Liu, 2009 ;  Hsueh, 2014 ).\nIn different subsets of tissues, PI3K is activated through Ras-dependent and Ras-independent signaling pathways ( Reichardt, 2006 ). In most neuronal cells, the Ras-dependent activation of PI3K initiates the major signaling pathways that produce several cell survival signals ( Vaillant  et al. , 1999 ). Additionally, the activation of PI3K can be induced through the Shc-Grb2-Gab1 (Grb2-associated binding protein-1) cascade. Specifically, this Ras-independent signaling pathway occurs when Shc is phosphorylated by TrkA and is associated with Grb2 to produce a complex, which further phosphorylates Gab1 adaptor protein and activates PI3K ( Holgado-Madruga  et al. , 1997 ). In addition, the phosphorylation of Trk receptors activates PI3K by promoting the phosphorylation of insulin receptor substrate-1 in some neurons ( Yamada  et al. , 1997 ). Once activated, either by a Ras-dependent or Ras-independent pathway, PI3K may phosphorylate several downstream effectors that are essential for NT-mediated cell function. Akt kinase is regarded as the most important intracellular protein that modulates the activity of certain transcription factors related to cell survival and apoptosis ( Brunet  et al. , 2001 ) (Fig.  2 ). One of the major regulators of cellular growth and metabolism activated by the PI3K/Akt downstream pathway is the mechanistic target rapamycin complex 1 or 2 (mTORC1 or mTORC2) ( Dibble and Cantley, 2015 ).\nAll NTs bind to p75NTR with relatively low affinity (Fig.  2 ). Following the binding of NTs to p75NTR, several signaling pathways that are mediated through the binding of p75NTR to several adaptor proteins may be activated ( Reichardt, 2006 ). One of the key pathways activated by NT-engaged p75NTR receptor binding is the Jun kinase signaling cascade, which leads to the activation of p53 and enhancement of cell apoptosis ( Reichardt, 2006 ) (Fig.  2 ). Additionally, the binding of NTs to p75NTR may induce the activation of nuclear factor-κB (NF-κB), which contributes to NF-κB-dependent neuronal survival ( Hamanoue  et al. , 1999 ) (Fig.  2 ). Furthermore, the interaction of NTs with p75NTR can activate acidic sphingomyelinase and induce the formation of ceramide ( Dobrowsky  et al. , 1994 ). Ceramide is responsible for the p75NTR-mediated amplification or inhibition effects on Trk-mediated cellular responses because ceramide modulates several signaling pathways, including ERK/MAPK, PI3K/Akt, Jun kinase and NF-κB, as well as the activity of the TrkA receptor in a cell type-dependent manner ( MacPhee and Barker, 1997 ;  Müller  et al. , 1998 ;  Zundel  et al. , 2000 ).\n\nSimilar to other members of the GDNF ligand family, GDNF signals through two functional receptors, GFRα1 and RET (for reviews, see  Takahashi, 2001 ). The GDNF homodimer first binds to GFRα1 (either a monomer or dimer), which then interacts with the extracellular domain of two RET molecules and induces their homodimerization and tyrosine autophosphorylation ( Airaksinen  et al. , 1999 ). Upon phosphorylation, the intracellular domain of the activated RET acts as high-affinity binding sites for several intracellular signaling proteins, including PI3K, MAPK and PLC-γ (for reviews, see  Airaksinen and Saarma, 2002 ). However, there is evidence for RET-independent signaling mechanisms, as many tissues express GDNF and GFRα1, but not RET ( Trupp  et al. , 1999 ). Recent studies have revealed that in the absence of RET, GDNF can interact with other receptors, such as neural cell adhesion molecules and integrins (for reviews, see  Fielder  et al. , 2018 ).\nThe GDNF signaling pathway may participate in crosstalk with other growth factors and receptors. In the peripheral nervous system, the endogenous TGF-β system is required as a cofactor for the exogenous GDNF-induced neuroprotective effect ( Airaksinen  et al. , 1999 ). Furthermore, TGF-β can induce cell responsiveness to GDNF by recruiting GFRα1 to the plasma membrane during several critical steps of signal transduction ( Peterziel  et al. , 2002 ). Similar co-operation has been shown between GDNF and NT signaling, as both GDNF and BDNF are required for tissue survival in sensory neurons ( Erickson  et al. , 2001 ). Indeed, a novel communication pathway between RET and NGF signaling has been demonstrated by using TrkA receptor ( Tsui-Pierchala  et al. , 2002 ). In the mammalian ovary, these synergistic, co-operative mechanisms seem to be of great importance for the development and maintenance of female reproductive function as these growth factors (TGF-β, NTs and GDNF), and their functional receptors are highly expressed in ovarian follicles ( Roy and Kole, 1998 ;  Abir  et al. , 2005 ;  Farhi  et al. , 2010 ).\n\nNTs and GDNF, as well as their cognate receptors, are expressed in preantral and antral follicles of the mammalian ovary. Additionally, data from clinical samples have shown that these neurotrophic factors are detectable in follicular fluid (FF;  Seifer  et al. , 2002 ,  2002 ;  Kawamura  et al. , 2008 ;  Palumbo  et al. , 2013 ). Furthermore, an imbalance of any of these intraovarian growth factors may contribute to abnormal follicular development and ovarian dysfunction or pathology in humans. The expression and localization of the NT and GDNF system in the mammalian ovary has been reviewed in detail elsewhere ( Streiter  et al. , 2016 ). In this review, we focused on the expression and potential function of these neurotrophic factors in the human ovary and their clinical connection to pathological conditions.\nIn the human ovary, primordial follicles undergo a series of histological and functional changes during follicular development. Under the stimulation of multiple intraovarian factors, the primordial follicle activates and changes into primary and, later, into secondary follicles, followed by transitions to tertiary (antral) follicles ( Hsueh  et al. , 2015 ). The development and growth of antral follicles is dependent on gonadotrophins, especially FSH ( Mihm and Bleach, 2003 ). To discuss the expression and localization of neurotrophic factors and their receptors in the human ovary, we divided the ovarian follicles into the preantral stage (primordial, primary and secondary) and antral stage (Table  II ).\n Table II Localization of neurotrophins/GDNF and receptors in the human ovary. Ligands/Receptors Localization Expression Detection method References NGF Preantral Follicles  Oocytes Protein IHC Abir  et al.  (2005)  GCs Protein IHC Abir  et al.  (2005) ,  Salas  et al.  (2006) Antral Follicles  GCs mRNA and Protein RT-qPCR, IHC Salas  et al.  (2006)  CCs Protein IHC Seifer  et al.  (2006)  TCs Protein IHC Salas  et al.  (2006)  FF Protein ELISA Palumbo  et al.  (2013) (213.76 pg/ml, ranged from  100 to 360 pg/ml) BDNF Preantral Follicles  Oocytes Protein IHC Harel  et al.  (2006)  GCs Protein IHC Harel  et al.  (2006) Antral Follicles  GCs mRNA and Protein RT-qPCR, IHC Zhao  et al.  (2011)  CCs Protein IHC Seifer  et al.  (2006)  FF Protein ELISA Seifer  et al.  (2002) (645.2 ± 23.6 pg/ml) a NT-3 Preantral Follicles  Oocytes Protein IHC Oron  et al.  (2011)  GCs Protein IHC Oron  et al.  (2011) Antral Follicles  GCs Protein IHC Seifer  et al.  (2006)  CCs Protein IHC Seifer  et al.  (2006)  FF Protein ELISA Seifer  et al.  (2002) (509 ± 97 pg/ml) a NT-4 Preantral Follicles  Oocytes mRNA and protein ISH, IHC Anderson  et al.  (2002) ,  Harel  et al.  (2006)  Pre-GCs mRNA and protein ISH, IHC Anderson  et al.  (2002)  GC Protein IHC Harel  et al.  (2006) Antral Follicles  CCs Protein IHC Seifer  et al.  (2006)  FF Protein ELISA Seifer  et al.  (2002) (397 ± 71 pg/ml) a TrkA Preantral Follicles  Oocytes Protein IHC Abir  et al.  (2005)  GCs Protein IHC Abir  et al.  (2005) ,  Salas  et al.  (2006) Antral Follicles  Oocytes Protein IHC Seifer  et al.  (2006)  GCs mRNA and Protein RT-qPCR, IHC Salas  et al.  (2006)  CCs Protein IHC Seifer  et al.  (2006)  TCs Protein IHC Salas  et al.  (2006) TrkB Preantral Follicles  Oocytes Protein IHC Anderson  et al.  (2002) ,  Harel  et al.  (2006)  Pre-GCs Protein IHC Anderson  et al.  (2002)  GC Protein IHC Harel  et al.  (2006) Antral Follicles  Oocytes Protein IHC Seifer  et al.  (2006)  CCs Protein IHC Seifer  et al.  (2006) TrkC Preantral Follicles  Oocytes mRNA and Protein ISH, IHC Oron  et al.  (2011)  GCs mRNA and Protein ISH, IHC Oron  et al.  (2011) Antral Follicles  Oocytes Protein IHC Seifer  et al.  (2006)  CCs Protein IHC Seifer  et al.  (2006) p75NTR Preantral Follicles  Stroma Protein IHC Anderson  et al.  (2002) ,  Abir  et al.  (2005) Antral Follicles  TCs Protein IHC Anesetti  et al.  (2001) GDNF Preantral Follicles  Oocytes Protein IHC Farhi  et al.  (2010)  GCs Protein IHC Farhi  et al.  (2010)  Stroma Protein IHC Farhi  et al.  (2010) Antral Follicles  Oocytes Protein IHC Farhi  et al.  (2010)  GCs mRNA and Protein RT-qPCR, IHC Farhi  et al.  (2010) ,  Zhao  et al.  (2011)  FF Protein ELISA Kawamura  et al.  (2008) (0.4–10.8 ng/ml) GFRα1 Preantral Follicles  Oocytes mRNA and Protein ISH, IHC Farhi  et al.  (2010)  GCs mRNA and Protein ISH, IHC Farhi  et al.  (2010)  Stroma Protein IHC Farhi  et al.  (2010) Antral Follicles  Oocytes Protein IHC Farhi  et al.  (2010)  GCs Protein IHC Farhi  et al.  (2010)  CCs mRNA RT-qPCR Cui  et al.  (2018) RET Preantral Follicles  Oocytes Protein IHC Farhi  et al.  (2010)  GCs Protein IHC Farhi  et al.  (2010)  Stroma Protein IHC Farhi  et al.  (2010) Antral Follicles  CCs mRNA RT-qPCR Cui  et al.  (2018) CCs, Cumulus cells; GCs, Granulosa cells; TCs, Theca cells; RT-qPCR, Quantitative real-time PCR; FF, Follicular fluid; ISH,  in situ  hybridization; IHC, Immunohistochemistry. a Data are expressed as the mean ± SD.\nLocalization of neurotrophins/GDNF and receptors in the human ovary.\nCCs, Cumulus cells; GCs, Granulosa cells; TCs, Theca cells; RT-qPCR, Quantitative real-time PCR; FF, Follicular fluid; ISH,  in situ  hybridization; IHC, Immunohistochemistry.\na Data are expressed as the mean ± SD.\nNGF mRNA transcripts and protein are expressed in both fetal and adult human ovaries; they are especially localized to the oocytes and GCs of preantral follicles (mainly from primordial to secondary follicles) ( Anderson  et al. , 2002 ;  Abir  et al. , 2005 ;  Salas  et al. , 2006 ) (Fig.  3 A). NGF protein has also been identified in both GCs (especially cumulus cells) and theca cells (TCs) of antral follicles ( Salas  et al. , 2006 ;  Seifer  et al. , 2006 ). Clinical samples obtained from infertile women undergoing IVF showed that NGF levels in FF are much higher than those in the serum (213.76 pg/ml vs. 46.47 pg/ml), indicating that NGF is locally produced in the ovarian follicle ( Palumbo  et al. , 2013 ) (Table  II ).\nImmunohistochemical staining of NGF, BDNF and GDNF in the human ovary . All sections were stained with the immunoperoxidase staining and then counterstained with Mayer’s haematoxylin.  (A)  Section of a human ovary from a 38-year-old woman. NGF is expressed in granulosa cells of the primordial follicle, and there is partial staining (with positive nuclear staining) in the oocyte. Original magnification X400. Reprinted with permissions from  Abir  et al.  (2005) .  (B)  Section of a human ovary from a 16-year-old girl. BDNF is expressed in the secondary (arrow) and primordial (arrow head) follicles. Original magnification X400. Reprinted with permissions from  Streiter  et al.  (2016) .  (C)  Section of a human ovary from a 22-year-old woman. GDNF is expressed in the oocytes and a portion of the granulosa cells and stroma cells. Original magnification X400. Reprinted with permissions from  Farhi  et al.  (2010) .\nBDNF is predominantly expressed in the somatic cells of primordial follicles in the human fetal ovary from 9 to 12 weeks gestation ( Childs  et al. , 2010 ). BDNF is also detected in the oocytes and GCs (only in a minority of samples) of fetal and adult ovaries ( Harel  et al. , 2006 ;  Streiter  et al. , 2016 ) (Fig.  3 B). Immunohistochemistry showed that BDNF was detected in cumulus cells (CCs) but at very low levels in the mural GCs of antral follicles ( Seifer  et al. , 2006 ;  Zhao  et al. , 2011 ;  Streiter  et al. , 2016 ) (Fig.  3 B). In mice, antral follicular levels of BDNF concomitantly increased with the LH surge during the preovulatory stage ( Kawamura  et al. , 2005 ). In humans, the administration of LH or hCG increased the secretion of BDNF (up to a 4- or 14-fold increase, respectively) in the conditioned medium of cultured CCs obtained from IVF patients, indicating that BDNF is produced by CCs and is positively regulated by gonadotrophins ( Feng  et al. , 2003 ). The mean FF (obtained from patients undergoing IVF) concentration of BDNF was 645.2 ± 23.6 pg/ml (Table  II ).\nTranscripts of NT-4 have been identified in the extracts of all ovarian tissues obtained from fetuses and women ( Harel  et al. , 2006 ). Similar to BDNF, NT-4 was primarily identified in oocytes and some GCs in 21-week-old fetal ovaries ( Harel  et al. , 2006 ). Likewise, NT-4 has been detected in FF (mean concentration: 397 ± 71 pg/ml) obtained from women undergoing IVF ( Seifer  et al. , 2002 ). NT-3 protein was identified in the oocytes and GCs of ovarian preantral follicles from fetuses, girls and women ( Oron  et al. , 2011 ). In the antral follicles, NT-3 protein was identified by immunohistochemistry in GCs and CCs ( Seifer  et al. , 2006 ), and the mature form of NT-3 protein was detected in the FF (mean concentration: 509 ± 97 pg/ml) ( Seifer  et al. , 2002 ) (Table  II ).\nIn the preantral follicles of the human ovary, TrkA protein has been detected mainly in oocytes, some stroma cells and GCs of ovarian samples obtained from fetuses, girls and women ( Abir  et al. , 2005 ). In the majority of fetal oocytes and oogonia TrkA was located in the cytoplasm but not in the nucleus ( Abir  et al. , 2005 ). Additionally, TrkA mRNA transcripts were identified in all fetal and adult ovaries examined ( Abir  et al. , 2005 ). Similarly, TrkB was mainly expressed in the oocytes/oogonia and some GCs of preantral follicles of the human fetal ovary ( Anderson  et al. , 2002 ;  Harel  et al. , 2006 ;  Childs  et al. , 2010 ). TrkC protein was expressed in the oocytes and GCs of preantral follicles in ovarian samples from fetuses (at 19–33 weeks of gestation), girls and women ( Oron  et al. , 2011 ). Using  in situ  hybridization, the same study showed that mRNA transcripts for full-length TrkC were detected in the oocytes of all samples examined and in GCs only in ovarian samples from girls and women ( Oron  et al. , 2011 ). The expression of p75NTR in mammals is species-dependent. In the preantral follicles of human ovaries, the mRNA and protein of pan-specific p75NTR were detected in stroma cells and TCs of ovarian samples from young fetuses (at 19–21 weeks of gestation) but not in those from older fetuses, girls and women ( Anesetti  et al. , 2001 ;  Abir  et al. , 2005 ).\nAll of the NT receptors, including TrkA, TrkB, TrkC and p75NTR, have been detected in the antral follicles of human ovaries ( Anesetti  et al. , 2001 ;  Seifer  et al. , 2006 ). Many studies have demonstrated the expression of TrkA in the major compartments of human antral follicles, including oocytes, GCs and TCs ( Shibayama and Koizumi, 1996 ;  Salas  et al. , 2006 ;  Seifer  et al. , 2006 ). Interestingly, the addition of hCG to cultured rat antral follicles rapidly increased the expression of TrkA, indicating that the increase in expression of TrkA during the preovulatory phase is LH dependent ( Dissen  et al. , 1995 ). In contrast to the expression pattern in rodents (in oocytes only), TrkB was detected in oocytes and CCs of antral follicles in humans ( Seifer  et al. , 2006 ). Similarly, TrkC protein has been identified in both oocytes and GCs in human antral follicles ( Shibayama and Koizumi, 1996 ;  Seifer  et al. , 2006 ). Using immunohistochemisty, p75NTR protein was identified in the TCs of human antral follicles ( Anesetti  et al. , 2001 ) (Table  II ).\nIn human preantral follicles, GDNF has been observed in the oocytes of all ovarian samples from fetuses and adult women, and in the GCs from half of all fetal samples and all adult samples ( Farhi  et al. , 2010 ) (Fig.  3 C). Additionally, GDNF mRNA was identified in human GCs of antral follicles and the expression can be upregulated by the addition of hCG alone or in combination with FSH ( Zhao  et al. , 2011 ). Moreover, mature GDNF protein was detectable in human FF and ranged from 0.4 to 10.8 ng/ml, indicating a paracrine function in the development of ovarian follicles ( Kawamura  et al. , 2008 ) (Table  II ).\nThe two functional receptors for GDNF, GFRα1 and RET, have been detected in the oocytes and GCs of human preantral and antral follicles ( Farhi  et al. , 2010 ). In particular, the mRNAs of GFRα1 and two RET isoforms were found in the oocytes and GCs of all ovarian samples from fetuses, girls and women. Our recent studies also showed that the mRNAs of GFRα1 and RET were detected in the CCs of mature follicles obtained from patients undergoing IVM ( Cui  et al. , 2018 ) (Table  II ).\n\nThe detection of NTs, GDNF and their cognate receptors in the principal cells (oocytes, GCs and TCs) throughout the different follicular stages of the human ovary may suggest that these intraovarian growth factors are involved in various stages of human folliculogenesis. Indeed, recent studies using animal models, clinical samples and  in vitro  cell culture have indicated that neurotrophic factors play indispensable roles in the central and peripheral nervous system, as well as in various tissues, including the ovary under normal and pathological conditions ( Streiter  et al. , 2016 ;  Skaper, 2018 ). Recent studies suggest an unexpected diversity of NT and GDNF actions in the regulation of ovarian and follicular development through nervous system stimuli and cell–cell interactions in the mammalian ovary ( Chaves  et al. , 2013 ;  Streiter  et al. , 2016 ) (Fig.  4 ).\nSchematic showing the neurotrophic factors involved in regulation of ovarian folliculogenesis . Neurotrophins, including NGF (in red), BDNF (in green), NT-3 (in purple), NT-4 (in yellow-brown), as well as GDNF (in blue), participate in and regulate follicular development at each of the defined stages throughout folliculogenesis, including follicle assembly, primordial follicle activation, follicular growth, steroidogenesis, oocyte maturation, ovulation and corpus luteum formation. Solid lines following each neurotrophic factor represent data obtained from studies in humans, while dashed lines represent data obtained from animal models.\nThe assembly of primordial follicles in the human ovary is a complex developmental process that involves the extensive co-ordination of many growth factors; among these factors, NTs are the major players ( Maheshwari and Fowler, 2008 ;  Grive and Freiman, 2015 ;  Streiter  et al. , 2016 ). The involvement of NTs in ovarian follicle assembly was supported by the identification of NTs and pan-specific p75NTR in the human fetal ovarian cortex during this process ( Anderson  et al. , 2002 ;  Abir  et al. , 2005 ). In particular, both NGF mRNA and protein are expressed in the human ovary prior to formation of the first primordial follicle ( Abir  et al. , 2005 ). NT-4 localizes to oocytes and pre-GCs in the fetal ovaries of humans from 13 weeks until the completion of gestation ( Anderson  et al. , 2002 ;  Harel  et al. , 2006 ). Furthermore, p75NTR mRNA and proteins were detected in the pre-GCs that surround oocytes only in fetal ovaries at gestational ages 13–21 weeks, representing the time before and during human follicle assembly ( Anderson  et al. , 2002 ;  Abir  et al. , 2005 ). Compared to the addition of neutralizing antibodies against NT-4, the addition of NT-4 to cultured human fetal ovaries enhanced follicle assembly  in vitro  ( Farhi  et al. , 2011 ). Animal studies also support a critical role of NTs in regulating ovarian follicle assembly. Depletion of both  Bdnf  and  Nt-4  in mice led to impaired follicular organization, increased oocyte death and grossly abnormal ovaries ( Ojeda  et al. , 2000 ;  Spears  et al. , 2003 ) (Table  III ). Mice lacking  Ngf  or  Trka  had a phenotype of reduced primordial follicle number ( Kerr  et al. , 2009 ) (Table  III ).  Trkb  knockout mice exhibited an increased number of non-encapsulated oocytes ( Kerr  et al. , 2009 ) (Table  III ). Similarly, the addition of NGF to the cultured ovaries of  Ngf -null mice resulted in an increased number of primordial follicles ( Lee  et al. , 1992 ;  Ojeda  et al. , 2000 ). Collectively, these results indicate that ovarian follicle assembly is most likely induced by the synergistic and complementary effects of these NTs and their corresponding receptors (Fig.  4 ).\n Table III Genetic mutations in the neurotrophin/GDNF system that cause ovary-specific phenotypes. Type of mutation Ovarian phenotype References NGF knockout mice Targeted deletion of the  Ngf  gene Reduced population of primordial, primary and secondary follicles, oocytes failed to be incorporated into follicular structure Dissen  et al.  (2001) NGF knockin mice Transgenic overexpression of the  Ngf  gene Reproductive and metabolic alterations characteristic of PCOS, arrested follicular development and increased apoptosis of antral follicles Wilson  et al.  (2014) ,  Dissen  et al.  (2009) NT-4 knockout mice Targeted depletion of the  Nt-4  gene Normal ovarian folliculogenesis Ojeda  et al.  (2000) Double NT-4/BDNF knockout mice Target depletion of both  Nt-4  and  Bdnf  gene Impaired follicular organization, increased oocyte death and grossly abnormal ovaries Ojeda  et al.  (2000) ,  Spears  et al.  (2003) GDNF knockout mice Targeted depletion of the  Gdnf  gene Reversal in the orientation of the ovary in relation to the abdominal viscera Moore  et al.  (1996) TrkA knockout mice Targeted depletion of the  Trka  gene Reduced number of primordial follicles, reduced number of FSH receptors Kerr  et al.  (2009) TrkB knockout mice Targeted depletion of the  Trkb  gene Reduced number of primordial follicles, reduced number of FSH receptors, an increased number of non-encapsulated oocytes Kerr  et al.  (2009) PCOS: polcystic ovary syndrome.\nGenetic mutations in the neurotrophin/GDNF system that cause ovary-specific phenotypes.\nPCOS: polcystic ovary syndrome.\nFollowing follicle assembly, the human ovary contains the maximum number of primordial follicles at birth (a few days after birth in rodents), which is defined as the ovarian reserve ( Findlay  et al. , 2015 ). These primordial follicles remain in a quiescent state in a primordial pool for decades until activation by certain stimuli in the surrounding microenvironment. Upon activation, the primordial follicle transforms into the primary follicle along with oocyte growth, TC recruitment and GC differentiation (from flat to cuboid shape) ( Kerr  et al. , 2013 ). Among the members of the NT family, NGF seems to be involved in the activation of primordial follicles as NGF is expressed in rat follicles at the primordial stage. Knockout mice lacking NGF had a reduced number of both primary and secondary follicles ( Dissen  et al. , 2001 ,  2002 ). Moreover, a functional study confirmed that the addition of NGF to cultured neonatal ovaries enhanced the activation rate of primordial follicles in mice ( Paredes  et al. , 2004 ). NT-3 activates the progression of primordial follicles to growing follicles in cultured rat ovaries ( Nilsson  et al. , 2009 ). NT-3 promotes the primordial to primary follicle transition by upregulating several target genes related to formation of the cytoskeleton and plasma membrane ( Nilsson  et al. , 2009 ). In sheep, GDNF improves the activation rate of primordial follicles in cultured ovaries ( Esmaielzadeh  et al. , 2013 ).\nRecent studies using transgenic mouse models have indicated that the intraovarian PI3K/Akt-mTOR pathway is the key signaling pathway that directs the transition from primordial to primary follicles as mice lacking several genes [v-akt murine thymoma viral oncogene homolog 1 ( Akt1 ), forkhead box O3 ( Foxo3 ), phosphatase and tensin homolog ( Pten ), ribosomal protein S6 ( RpS6 ) or tuberous sclerosis complex 1/tuberous sclerosis complex 2  (Tsc1 / Tsc2 )] related to this pathway had a phenotype of premature depletion of primordial follicles ( Adhikari and Liu, 2009 ). A recent study using a rodent model demonstrated that localized ovarian trauma or ovulation can induce the activation of primordial follicles, which was completely blocked by the mTOR inhibitor rapamycin ( He  et al. , 2017 ). A trauma-induced transient increase in locally produced NGF was proposed to trigger the activation of dormant primordial follicles through the PI3K/Akt-mTOR signaling pathway from pre-GCs to oocytes ( He  et al. , 2017 ). This proposal may provide a molecular mechanism by which the signaling changes in the surrounding intraovarian environment affect the selective recruitment of a cohort of primordial follicles during the menstrual period. Indeed, the injury-induced activation and development of follicles can be used to explain the beneficial effects of ovarian wedge resection and laparoscopic ovarian diathermy drilling for treating women with polycystic ovary syndrome (PCOS;  Lebbi  et al. , 2015 ).\nNTs are crucial growth factors for the survival, maintenance, and development of neurons in the central and peripheral nervous system ( Huang and Reichardt, 2001 ). These neuronal growth factors also provide sympathetic innervation and trophic support for the ovary, which is essential for follicular development ( Dissen  et al. , 2002 ). In humans, the addition of Trk receptor blockers to fetal ovaries  in vitro  (obtained at early gestation: 13–16 weeks) decreased oogonia survival, indicating an important role for NTs in maintaining germ cell survival ( Spears  et al. , 2003 ).  In vitro  studies have demonstrated that treatment with NGF decreased cell apoptosis and increased the follicular growth of mouse ovaries by downregulating apoptosis-related genes ( Roh and Pi, 2013 ). Additionally, NGF promoted follicular development and increased cell responses to FSH (and the formation of cAMP) by upregulating FSH receptors in neonatal rat ovaries, indicating crosstalk between this NT and the endocrine signaling system ( Romero  et al. , 2002 ). Similarly, NGF also upregulated the expression of the FSH receptor in human GCs  in vitro  ( Salas  et al. , 2006 ). In addition to the effects on germ cells and GCs, NGF has been shown to participate in promoting TC proliferation. In an  in vitro  culture of TCs from antral follicles, transfection with TrkA-expressing plasmids in the presence of NGF resulted in an increase in the bovine TC proliferation rate ( Dissen  et al. , 2000 ). The addition of NT-3 to the culture medium also increased the cell proliferation rate in bovine TCs  in vitro  ( Dissen  et al. , 2000 ).\nGDNF and its cognate receptors are expressed in human fetal and adult ovaries and promote follicular development in rat ovaries ( Dole  et al. , 2008 ;  Farhi  et al. , 2010 ). Rat ovaries cultured in the presence of GDNF showed an increased number of developing follicles without an increased total number of ovarian follicles ( Dole  et al. , 2008 ). Additionally, the combined treatment of cultured ovaries with GDNF, epidermal growth factor (EGF), kit ligand, and fibroblast growth factor enhanced the differentiation and viability of primordial follicles in sheep ( Esmaielzadeh  et al. , 2013 ). Moreover, the most recent studies have shown that GDNF inhibited cell apoptosis in human CCs  in vitro  ( Cui  et al. , 2018 ).\nOvarian steroidogenesis products are the key components for maintaining normal ovarian function, including follicular development, oocyte maturation, ovulation and embryo implantation ( Chang  et al. , 2016a ). In mature human follicles, NGF is apparently involved in the modulation of steroid hormone production. For instance, NGF inhibited the differentiation of GCs into luteal cells by suppressing the production of progesterone in isolated human GCs ( Salas  et al. , 2006 ). Alternatively, NGF directly promoted the production of estradiol and indirectly enhanced the cell response to FSH (FSH increases the production of estradiol in GCs) by upregulating expression of the FSH receptor in human GCs, which occurred through the activation of high-affinity tyrosine kinase NGF receptor TrkA ( Romero  et al. , 2002 ;  Salas  et al. , 2006 ). In contrast, the addition of antibodies blocking NGF reversed the inducing effect of NGF on estradiol secretion ( Salas  et al. , 2006 ). In bovine antral follicles, NGF increased the production of both androgen and progesterone in freshly plated TCs through the TrkA receptor-mediated signaling pathway ( Dissen  et al. , 2000 ). NT-3 is another intragonadal molecule that is involved in promoting follicular steroidogenesis ( Waraksa  et al. , 1995 ). When NT-3 (at 100 ng/ml but not higher doses) was added to hamster follicles  in vitro , NT-3 induced a two-fold increase in estradiol production ( Waraksa  et al. , 1995 ).\nOocyte maturation is a critical reproductive event that involves a series of functional and morphological changes, including nuclear maturation, cytoplasmic reorganization, meiotic resumption, cytoskeletal dynamics and gamete-somatic cell interactions ( Coticchio  et al. , 2015 ). Oocyte nuclear maturation is characterized by germinal vesicle breakdown and extrusion of the first polar body (PBI), whereas oocyte cytoplasmic maturation is characterized by cytoplasmic changes essential for monospermic fertilization, and the subsequent preparation for development into preimplantation embryos ( Eppig, 1996 ).\nIn addition to the endocrine system driven by the hypothalamic-pituitary-ovarian axis, oocyte maturation relies on several locally produced intraovarian factors ( Hsueh  et al. , 2015 ;  Chang  et al. , 2016b ). NTs (especially BDNF and NT-4) and GDNF appear to be the crucial intrafollicular molecules that play a supporting role in oocyte maturation. In mice, oocytes cultured in the presence of BDNF showed increased oocyte maturation and PBI extrusion (an indicator of oocyte nuclear maturation) rates, which improved the competence of oocytes to complete preimplantation development ( Seifer  et al. , 2002 ;  Kawamura  et al. , 2005 ). Similarly,  in vitro  culture of mouse ovaries with a high concentration (100 ng/ml) of NT-4 resulted in an increased rate of PBI extrusion ( Seifer  et al. , 2002 ). In humans, BDNF FF levels obtained from women undergoing IVF are positively correlated with the number of mature oocytes ( Wang  et al. , 2011 ). Additionally, combined treatment with BDNF, EGF and insulin-like growth factor-1 (IGF-1) improved the maturation rates of human oocytes and subsequent embryo development ( Yu  et al. , 2012 ).\nGDNF has been identified in the GCs/CCs of antral follicles in several species, including mice, pigs and humans ( Linher  et al. , 2007 ;  Dole  et al. , 2008 ;  Zhao  et al. , 2011 ). In addition, the mature form of GDNF protein was highly detectable in porcine and human FF ( Linher  et al. , 2007 ;  Zhao  et al. , 2011 ). Animal studies using porcine oocytes have shown that GDNF significantly enhanced CC expansion and increased the extrusion of the PBI ( Linher  et al. , 2007 ). In humans, functional studies have demonstrated the role of GDNF in enhancing the developmental competence of oocytes ( Zhao  et al. , 2011 ). Consistent with these results, our recent studies showed that exogenous GDNF promoted the maturation  in vitro  of cultured immature oocytes to metaphase II (MII) stage ( Cui  et al. , 2018 ). Human microRNA (miRNA) analyses revealed that the addition of GDNF to culture medium induced several differentially downregulated miRNAs among in vitro matured CCs, which most likely mediated GDNF-induced maintenance of CC viability and enhancement of oocyte maturation ( Cui  et al. , 2018 ). Intriguingly, mice lacking  Gdnf  had an abnormal ovarian topology that these transgenic mice exhibited a reversal in orientation of the ovary in relation to their abdominal viscera ( Moore  et al. , 1996 ) (Table  III ).\nBased on the mechanisms that initiate the ovulatory cascade, mammals are classified as either spontaneous (e.g. mice, pigs, horses and cattle) or induced (e.g. cats, rabbits, koala and camelids) ovulators ( Silva  et al. , 2015 ). In spontaneous ovulators, ovulation is regularly triggered by an increase in the circulating concentration of estradiol, whereas in induced ovulators, ovulation is induced by stimulation of the female genital tract during the copulation ( Silva  et al. , 2015 ). Animal studies have provided evidence that NGF is involved in the process of mammalian ovulation. In rats, both NGF and Trka receptor are dramatically increased in the cells of the follicular wall and interstitial tissue of the ovary, reaching their peak levels ~5 h before ovulation ( Dissen  et al. , 1996 ).  In vivo  administration of antibodies against NGF attenuated the release of ovarian prostaglandin E 2  and subsequently suppressed ovulation, indicating the role of NGF in triggering rodent ovulation ( Dissen  et al. , 1996 ). Furthermore, NGF-induced activation of TrkA receptors can disrupt cell–cell communication by affecting the integrity of the connexin 43-based gap junctions, which further induced cellular dissociation of the follicular wall that precedes ovulatory rupture in rats ( Mayerhofer  et al. , 1996 ).\nNGF (especially β-NGF) has been regarded as the ovulation-inducing factor that is present in the seminal plasma of some male mammals ( Ratto  et al. , 2012 ). Indeed, intramuscular, intravenous or intrauterine administration of NGF consistently induced ovulation in a dose-dependent manner in several induced ovulatory mammals, such as llamas, camelids and alpacas ( Ratto  et al. , 2005 ;  Silva  et al. , 2015 ;  El Allali  et al. , 2017 ). A recent study using a camel model proposed that NGF in the seminal plasma was absorbed through the endometrium to the bloodstream and transferred to the hypothalamus where this growth factor stimulates neurons to produce kisspeptin (a potent stimulator of GnRH), which eventually regulates the preovulatory LH surge ( El Allali  et al. , 2017 ). At present, the functional role of NTs in the regulation of ovulation in humans remains unknown.\nIn addition to the functional role of NGF in regulating ovulation, NGF was associated with enhanced tissue vascularization during the early stage of corpus luteum development in llamas ( Ulloa-Leal  et al. , 2014 ). Several studies using animal models have revealed that the preovulatory LH surge induced by NGF can be sustained remarkably longer than that induced by GnRH administration, indicating that the luteotrophic effect is most likely triggered by NGF due to prolonged LH secretion ( Adams  et al. , 2005 ;  Silva  et al. , 2011 ;  Ulloa-Leal  et al. , 2014 ). Subsequent studies have confirmed the luteotrophic role of NGF in regulating corpus luteum function through direct or indirect effects on GnRH neurons in the hypothalamus ( Silva  et al. , 2011 ;  Fernández  et al. , 2014 ;  El Allali  et al. , 2017 ).\n\nGiven the important roles for NTs in regulating follicular development and oocyte competence, we can speculate that dysregulation of the NT system may negatively affect ovarian function, leading to reproductive pathology or female infertility. Clinical data obtained from fertile women revealed a cyclic change in plasma BDNF levels, which were positively correlated with estradiol and progesterone and negatively correlated with age ( Begliuomini  et al. , 2007 ;  Pluchino  et al. , 2009 ). Additionally, the concentrations of BDNF steadily declined after menopause ( Begliuomini  et al. , 2007 ). Interestingly, in menopausal women, hormone replacement therapy restored their plasma BDNF to levels comparable to those in premenopausal women ( Begliuomini  et al. , 2007 ). In mice, chronic unpredictable stress decreased the expression of BDNF in the antral follicles and reduced the number of mature oocytes and formation of blastocyst embryos, which was reversed by exogenous treatment with BDNF ( Wu  et al. , 2012 ). In three goat breeds, a single nucleotide polymorphism (SNP; A705G) that was associated with larger litter size was detected in the NGF gene, indicating that NGF could potentially be used as a marker for goat litter size ( An  et al. , 2013 ).\nAltered FF levels of NTs have been identified in women with different etiologies of infertility undergoing IVF treatment, indicating the association of dysregulated NTs with infertility ( Buyuk and Seifer, 2008 ). Compared to women with normal ovarian function, women with diminished ovarian reserve have higher FF levels of NGF and lower (although not statistically significant) FF levels of BDNF ( Buyuk and Seifer, 2008 ). Lower expression levels of BDNF in women with diminished ovarian reserve may reflect their ovarian ages and a gradual decline in follicle quantity. Although many studies have demonstrated the decline in BDNF levels with advancing chronological age in women ( Begliuomini  et al. , 2007 ;  Ziegenhorn  et al. , 2007 ;  Buyuk and Seifer, 2008 ), BDNF (unlike anti-Müllerian hormone) does not reflect ovarian function or serve as an indicator for cancer in women receiving chemotherapy ( Aslam  et al. , 2011 ).\nIn humans, NGF levels were significantly higher (~4- to 5-fold) in FF than in serum, indicating local production of NGF in the ovarian follicle ( Palumbo  et al. , 2013 ). Since NGF is expressed in the oocytes and GCs from primordial to secondary follicles in human fetal and adult ovaries, higher expression levels of NGF may accelerate the activation of primordial follicles that eventually leads to poor ovarian reserve. Another study using clinical samples obtained from women undergoing IVF treatment also showed that an increased FF level of NGF was correlated with a lower ovary response (especially in older women) ( Palumbo  et al. , 2013 ). However, the high expression levels of NGF may be the byproduct of a lower response of germ cells or follicular cells to NGF receptors. Indeed, an  in vitro  study demonstrated that decreased expression levels of the NT receptors TrkA and p75NTR in human CCs were associated with diminished ovarian reserve in women undergoing ART ( Buyuk  et al. , 2011 ). At present, studies using genetic approaches have not detected any naturally occurring mutations in genes of NTs or their receptors that are associated with women with primary ovarian insufficiency.\nNGF and BDNF are two neurotrophic factors that are most likely involved in the pathogenesis of PCOS. The BDNF levels in both FF and plasma were higher in women with PCOS than in healthy women ( Russo  et al. , 2012 ). In human GCs, the addition of hCG to culture medium increased the levels of BDNF up to 3- to 4-fold, indicating that locally produced BDNF is mainly regulated by LH or hCG ( Feng  et al. , 2003 ;  Zhao  et al. , 2011 ). Therefore, the high levels of LH in women with PCOS likely contribute to increased expression of BDNF in GCs, resulting in increased secretion of BDNF in the FF and plasma ( Russo  et al. , 2012 ). These findings suggest that BDNF could be a candidate neurotrophic factor capable of promoting follicular development. Indeed, the levels of BDNF in FF and plasma have been identified as potential predictors of IVF outcomes ( Monteleone  et al. , 2007 ;  Wang  et al. , 2011 ).\nIn rats, exogenous administration of steroids induced an increase in intraovarian synthesis of NGF and p75NTR, which led to polycystic ovaries that were characterized by the appearance of multiple follicular cysts ( Lara  et al. , 2000 ). In mice, transgenic overexpression of  Ngf  targeted to the ovary caused an ovarian cystic morphology that represented both the reproductive and metabolic features of human PCOS ( Dissen  et al. , 2009 ;  Wilson  et al. , 2014 ) (Table  III ). Morphological evaluation of the ovaries in these mice showed that overproduction of NGF led to arrested follicular development and increased apoptosis of antral follicles ( Dissen  et al. , 2009 ). Collectively, these findings support the hypothesis that sympathetic neuron hyperactivity of the ovary may contribute to the development and progression of PCOS ( Wilson  et al. , 2014 ). Indeed, clinical data obtained from patients undergoing IVF revealed that women with PCOS had an approximate 2-fold increase in the FF level of NGF and a 6-fold increase in NGF expression in GCs compared with those in women without PCOS ( Dissen  et al. , 2009 ). In contrast, another clinical study did not show a similar result regarding the increased FF levels of NGF in patients with PCOS ( Buyuk and Seifer, 2008 ).\nAffecting ~10% of reproductive-aged women, endometriosis is a chronic gynecological disease that is defined as the appearance of endometrial fragments outside the uterine cavity ( Vigano  et al. , 2004 ). Despite extensive investigations, the precise pathogenesis and detailed molecular mechanisms of this common disease remain elusive ( Bulun  et al. , 2015 ). Emerging evidence has suggested a critical role for NTs in uterine physiology and endometrial pathology ( Barcena de Arellano  et al. , 2013 ;  Wessels  et al. , 2014 ). The immunointensity of NGF and TrkA was higher in both the endometriotic stroma and epithelium obtained from women with endometriosis and deep dyspareunia than that in those obtained from women with endometriosis without deep dyspareunia, indicating that NGF signaling may play an important role in endometriosis-associated sexual pain ( Peng  et al. , 2018 ). A clinical study demonstrated that women with endometriosis had higher levels of NGF in their FF than did patients with male factor infertility ( Buyuk and Seifer, 2008 ).\nProteomic analyses have identified the presence of both BDNF and NT-4 in the eutopic endometrium of women with endometriosis ( Browne  et al. , 2012 ). Additionally, estrogen receptor, BDNF and TrkB mRNA and protein were detected in the ectopic endometrium of women with ovarian endometriosis ( Yu  et al. , 2016 ). Furthermore, the estrogen-induced upregulation of BDNF and TrkB that enhances the proliferation of endometrial cells in uterine tissue has been proposed to explain the pathogenesis of estrogen-dependent diseases, including endometriosis ( Wessels  et al. , 2015 ;  Dong  et al. , 2017 ). Indeed, recent clinical studies have reported that plasma concentrations of BDNF were significantly higher in women with endometriosis, especially in Stages I and II disease, indicating that BDNF is a potential clinical marker for this painful disorder during its early stage ( Wessels  et al. , 2016 ;  Rocha  et al. , 2017 ;  Perricos  et al. , 2018 ). Targeted genetic association analysis of a case-control study including 425 patients with endometriosis revealed the presence of a BDNF SNP that was linked to an increased severity of endometriosis (Stage III and Stage IV) ( Zhang  et al. , 2012 ). Interestingly, higher FF levels of BDNF were detected in women with unexplained infertility than in those with known causes of infertility ( Sadeu  et al. , 2012 ). Future studies will be of great interest to determine whether NTs are involved in the pathogenesis of endometriosis or whether they are merely disease-related byproducts.\nEpithelial ovarian cancer (EOC) represents approximately 90% of total ovarian cancer cases; EOC is a deadly cancer type with a low 5-year survival rate ( Siegel  et al. , 2011 ). In mammals, NGF and its receptor TrkA might be involved in the regulation of morphological changes in ovarian surface epithelium during the breeding season ( Bao  et al. , 2014 ). In humans, NGF and TrkA are highly expressed in EOC and are associated with the processes that are crucial for EOC development, including cancer cell proliferation, migration, invasion and angiogenesis ( Vera  et al. , 2014 ). TrkA-mediated NGF-induced angiogenesis in endothelial cells promotes the development and progression of EOC ( Julio-Pieper  et al. , 2006 ;  Vera  et al. , 2014 ).  In vitro  studies have demonstrated that NGF upregulated the expression of VEGF, which contributes to the metastatic potential of a tumor ( Julio-Pieper  et al. , 2006 ;  Tapia  et al. , 2011 ). In addition to VEGF, other molecules regulated by NGF that are involved in the crucial process required for the progression of EOC include calreticulin (CRT), cyclo-oxygenase-2, disintegrin, metalloproteinase domain-containing protein 17 and several miRNAs (for reviews see ( Retamales-Ortega  et al. , 2017 )). These studies suggest that NGF may be applied as a biomarker for ovarian cancer.\n\nIn many mammals, including humans, NGF, BDNF and GDNF are associated with oocyte maturation and embryo development ( Seifer  et al. , 2002 ;  Kawamura  et al. , 2005 ;  Wang  et al. , 2011 ;  Linher-Melville and Li, 2013 ;  Cui  et al. , 2018 ). In clinical application, both FF and plasma levels of BDNF are reportedly potential predictors of pregnancy outcome during IVF/ICSI treatment ( Monteleone  et al. , 2007 ;  Wang  et al. , 2011 ). Furthermore, the addition of BDNF and GDNF to cultured immature human oocytes and human CCs promoted total yields of MII oocytes and CC viability, respectively ( Zhao  et al. , 2011 ;  Cui  et al. , 2018 ). Similarly, combined treatment with BDNF, EGF and IGF-1 improved the maturation rates of human oocytes and subsequent embryo development ( Yu  et al. , 2012 ). Collectively, these findings have provided insights into the roles of NTs and GDNF in the regulation of oocyte maturation and CC viability. Supplementation of the IVM culture medium with these intraovarian growth factors (alone or in combination) may eventually be applied to enhance the developmental potential of immature oocytes obtained from patients undergoing ART.\nGiven the indispensable role of NGF-induced PI3K/Akt-mTOR signaling in the activation of dormant primordial follicles, a potential novel infertility therapy has been proposed by using PTEN inhibitors and PI3K stimulators to promote the PI3K/Akt-mTOR-mediated activation of residual follicles in patients with primary ovarian insufficiency ( Kawamura  et al. , 2013 ). Based on animal studies showing that treatment with the mTOR inhibitor rapamycin can preserve the size of rat follicles ( Adhikari  et al. , 2013 ), we speculate that blocking agents targeting ovary-specific mTOR signaling or NGF function could be a good option in the future for fertility preservation in women, who now have an extended life expectancy. However, future studies on NGF and human primordial follicle activation are needed.\nData obtained from clinical samples showing that plasma concentrations of BDNF were significantly higher in women with endometriosis (Stages I and II) ( Wessels  et al. , 2016 ;  Rocha  et al. , 2017 ;  Perricos  et al. , 2018 ) may indicate the potential use of BDNF in diagnosing this chronic disease in its early stage. The participation of NGF signaling in the development of ovarian cancer and the findings that NGF and TrkA are highly expressed in women with EOC suggest that NGF may be applied as a biomarker for ovarian cancer. In human ovarian surface epithelium and EOC cell lines, NGF increased the expression of chaperone protein CRT, which was reversed by the addition of a specific TrkA receptor inhibitor ( Vera  et al. , 2012 ), suggesting that blocking NGF activity could potentially be used as a therapeutic approach or adjuvant therapy in treating patients with EOC.\n\nRecently, there has been substantial interest in locally produced growth factors and intrafollicular signaling between the oocyte and its supporting follicle cells. Evidence supported by animal studies and clinical trials in humans has suggested that intraovarian NTs and GDNF play crucial roles in regulating ovarian function, ranging from ovarian follicle assembly, primordial follicle activation, follicular growth and development, to steroidogenesis, oocyte maturation, ovulation and corpus luteum formation (Fig.  4 ). Normal ovarian function and follicular development rely on the synergistic and complementary effects of these neurotrophic factors and their corresponding receptors. Dysregulation of these intraovarian growth factors may lead to the development of infertility or ovarian diseases, such as diminished ovarian reserve, PCOS, endometriosis and even ovarian cancer. As our understanding of the expression, actions and underlying molecular mechanisms of these neurotrophic factors in the human ovary expands, the advancement of new diagnostic and therapeutic applications for the management of patients with infertility and ovarian pathology, as well as improvement in oocyte quality, will become plausible.\n\nH.M.C. collected the information, designed the pictures and wrote the manuscript. H.C.W. collected the information and designed the pictures. Z.G.S. collected the information and wrote the manuscript. F.L collected the information and critically revised the manuscript. P.C.K.L. collected the information, designed the pictures and critically revised the manuscript. All the authors have seen and approved the final version.\n\nThis work was supported by the Foundation Scheme Grant FDN-143317 from the Canadian Institutes of Health Research to P.C.K.L. This work was also supported by the National Natural Science Foundation of China (Grant no. 81774355) to F.L.\n\nWe declare that H.C., H.W., Z.S., F.L. and P.C.L. have no conflict of interest with the contents of this manuscript.","source_license":"public-domain-us","license_restricted":false}