- Email: [email protected]
Immunocytochemical evidence for the presence and location of the neurotrophin–Trk receptor family in adult human preovulatory ovarian follicles
American Journal of Obstetrics and Gynecology (2006) 194, 1129–36
www.ajog.org
Immunocytochemical evidence for the presence and location of the neurotrophin–Trk receptor family in adult human preovulatory ovarian follicles David B. Seifer, MD,* Bo Feng, PhD, Robert M. Shelden, PhD Department of Obstetrics, Gynecology and Reproductive Sciences, Division of Reproductive Endocrinology and Infertility, UMDNJ-Robert Wood Johnson Medical School, New Brunswick, NJ Received for publication November 14, 2005; accepted December 15, 2005
KEY WORDS Neurotrophins Trk receptors Granulosa Oocytes Follicles Growth factors Ovary
Objective: This study was undertaken to evaluate the presence or absence of neurotrophins and their respective receptors within adult human preovulatory follicles. Study design: Prospective study of neurotrophins and their receptors in follicular cells and unfertilized oocytes from women undergoing aspiration for in vitro fertilization/intracytoplasmic sperm injection. Cells (mural and cumulus granulosa cells, unfertilized oocytes) were examined for immunocytochemical staining of neurotrophin and receptor proteins. Results: Mural and cumulus granulosa cells were positive for BDNF, NT-4/5, NT-3, and NGF, as well as for Trk B, Trk C, and Trk A receptors. Unfertilized oocytes were positive for Trk B, Trk C, and Trk A receptors. Conclusion: Neurotrophins and their respective receptor proteins are present within the mural and cumulus granulosa cells of adult human preovulatory follicles. Neurotrophin receptors are present in human unfertilized oocytes. The location of the neurotrophins and their receptors suggest both an autocrine and paracrine function within the adult human ovarian follicle. Ó 2006 Mosby, Inc. All rights reserved.
Development and maturation of vertebrate gametes is the culmination of a highly complex series of processes involving nonovarian (eg, hypothalamic releasing/stimulating factors, hypophyseal gonadotropin hormones), ovarian (steroid hormones), and cellular (cell signaling)
Supported in part by the Samuel A. Pasquale grant. Presented at the Twenty-Fourth Annual Meeting of the American Gynecological and Obstetrical Society, September 29-October 1, 2005, Victoria, British Columbia, Canada. * Reprint requests: David B. Seifer, MD, Maimonides Medical Center, 1355 84th St, Brooklyn, NY 11228. E-mail: [email protected] 0002-9378/$ - see front matter Ó 2006 Mosby, Inc. All rights reserved. doi:10.1016/j.ajog.2005.12.022
inputs exquisitely orchestrated to produce gametes capable of fertilization. The oocyte carries the additional requirement for storing sufficient information and resources to support the newly formed zygote through 2 or more cell divisions until the newly emerging embryo genome is able to assume command of subsequent development.1,2 Although much remains to be elucidated, endocrine regulation of the hypothalamic-hypophyseal-gonadal axis through positive and negative feedback loops has been well established. Intragonadal and intracellular regulatory pathways and mechanisms are less well appreciated, but significant gains in understanding of
1130 these processes have occurred. It is, for example, well established through the work of Eppig and others3-5 that the oocyte not only receives information and material from the surrounding granulosa cells (corona radiata), but also plays a highly significant reciprocal role in regulating the activity and production of these cells. Moreover, it is clear that a multitude of growth factors, eg, growth and development factor-9 (GDF-9), insulinlike growth factor (IGF), transforming growth factor (TGF-ß), bone morphogenetic protein (BMP)6 and adhesion factors, eg, connexins,7 are critical in the developmental processes involved in the intrafollicular oocyte-somatic cell communication network that leads to mature oocytes. More recently, it has been recognized that neurotrophins, a family of soluble polypeptide growth factors well known for their role in neuronal survival and neural outgrowth, may be intimately involved in the dynamic development and maturation of the ovarian follicle/ oocyte complex. Although other neurotrophic factors have been identified, the neurotrophins found in a variety of non-neuronal systems (eg, cardiovascular, immune, endocrine, and reproductive systems8,9) include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-4/5 (NT-4/5), and neurotrophin-3 (NT-3). BDNF, NT-4/5, NT-3, and NGF and their respective tyrosine kinase (Trk) receptors (Trk B for BDNF and NT-4/5, Trk C for NT-3, and Trk A for NGF) have been identified in the mammalian ovary10 and have been shown to play a role in ovulation,11,12 steroid secretion,13 and follicular development14-16 in the rodent. Recently, we described presumptive evidence for the secretion of neurotrophins and the presence of their receptors in human cumulus cells.17-20 We first reported the presence of BDNF in the follicular fluid of the human ovarian follicle. We demonstrated its secretion by human cumulus granulosa cells and its promotion of mouse oocyte maturation presumably via its preferred receptor, Trk B.17 We reported the presence of NT-4/5, also exhibiting preferential binding to the Trk B receptor, and NT-3, a non-Trk B ligand within the human ovarian follicle.18 We further demonstrated the presence of Trk B receptor in the immature mouse oocyte and BDNF-induced acceleration of first polar body formation by cultured immature mouse oocytes.17,18 It is emphasized that these prior studies examined the majority of neurotrophins secreted in specifically follicular fluid and cell culture media, not directly by cells per se, using commercially available enzyme-linked immunosorbent assays (ELISAs) with investigation of neurotrophin receptor presence limited to only the examination of mouse oocytes.17,18 In the current study, we extended these investigations to include direct immunocytochemical evidence of neurotrophins and their respective receptors in the basic
Seifer, Feng, and Shelden cellular components of the human follicle that include mural granulosa cells, cumulus granulosa cells, and oocytes obtained from women undergoing oocyte aspiration for in vitro fertilization. Our objective was to determine whether neurotrophins and their respective receptors are present within the adult human preovulatory follicles of women undergoing follicular aspiration for in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI).
Material and methods Population Follicular fluid was obtained from 29 women younger than 42 years undergoing follicular aspiration for IVF/ ICSI. Women had received either leuprolide acetate (Lupron, TAP Pharmaceuticals, Inc, North Chicago, IL) for pituitary desensitization, followed by 2 to 8 ampules of gonadotropins given daily in divided morning and evening doses as previously described21 or daily gonadotropins until a lead follicle was 14 mm or greater in mean diameter whereupon cetrorelix acetate (Cetrotide, Serono, Norwell, MA) was added to prevent premature ovulation. Transvaginal follicular aspiration was performed under sedation 36 hours after administration of 5,000 or 10,000 IU hCG when at least 4 lead follicles were 18 mm or greater in mean diameter. The UMDNJRobert Wood Johnson Medical School Institutional Review Board approved this study protocol.
Human mural and cumulus granulosa cell culture Pooled clear follicular fluid was centrifuged 10 minutes, 325!g at 20(C. The top layer of cells enriched with mural granulosa was collected into Hanks’ Balanced Salt Solution (HBSS, Ca and Mg-free, Sigma, St Louis, MO) then overlaid on top of 45% colloidal silica solution (Enhance-S Plus, Conception Technologies, San Diego, CA) and centrifuged for 20 minutes at 325!g. After separation, the mural granulosa cell enriched interface layer was collected and washed in HBSS. The resultant mural granulosa cell pellet was incubated in collagenase type IA (100 m/mL, Sigma) at room temperature until cell clumps separated. Cells were washed twice then plated in 8-chamber slides (Nalge Nunc International Corp, Naperville, IL). Cumulus granulosa cells were removed from oocytes in preparation for IVF/ICSI and then were incubated with hyaluronidase (100 IU/mL, Sigma), followed by 2! wash with HBSS. For each experiment, cumulus cells from 2 to 3 patients were pooled, then plated onto 8-chamber slides. Both cell types (mural and cumulus granulosa) were cultured in Ham’s F-10 medium supplemented with 10% fetal bovine serum (FBS) at 37(C, 5% CO2 in air. Each experiment was carried out a minimum of 3 times.
Seifer, Feng, and Shelden
Human oocytes Ninety-eight human unfertilized oocytes were obtained day 0 to day 2 from 31 consenting patients undergoing IVF/ICSI after retrieval on day 0. Day 0 oocytes were immature (germinal vesicle or Meiosis I stage), whereas day 1 and day 2 oocytes were mature (first polar body present) on day 0 but did not exhibit pronuclei after insemination or injection. Isolated oocytes were washed twice with phosphate-buffered saline solution (PBS) and were fixed with 4% paraformaldehyde for 10 minutes, followed by PBS wash. Fixed oocytes were stored at 4(C in blocking buffer (1.5% normal goat serum in PBS) until used for immunocytochemistry procedures described below. Oocyte (day 0-2) Trk staining was repeated in at least 3 independent staining procedures. Photographs were taken immediately after the final wash using an Imagine Photographer (Scanalytics, Inc, Fairfax, VA).
Immunocytochemistry staining After an initial 24-hour culture, granulosa cells were washed twice with PBS then fixed with 4% paraformaldehyde for 10 minutes, followed by 3! wash with PBS. Immunocytochemistry staining of neurotrophins and their receptors was carried out at room temperature using ABC reagent (Vectastain Elite, Vector Laboratories, Burlingame, CA). In brief, fixed cells or oocytes were treated with blocking serum (1.5% goat serum with 0.3% Triton-X100 in PBS). After washing with PBS, cells or oocytes were incubated overnight at 4(C with primary antibody against BDNF (Promega, Madison, WI), NT-3, NT-4/5, NGF, Trk A, Trk B, Trk C (Santa Cruz Biotechnology, Inc, Santa Cruz, CA). Antibodies used in these studies were affinity purified polyclonal rabbit antibodies that demonstrated no cross-reactivity with related Trk or neurotrophin species. Trk A antibody (sc-118) was directed against a carboxy terminus region of the gp140 precursor form of human Trk A. Trk B antibody (sc-12) was directed against a carboxy terminus region of the human Trk B precursor gp145. The Trk B antibody binds to Trk B of mouse, rat, and human origin but does not bind to the truncated form of Trk B. Trk C (sc-117) antibody was directed against a carboxy terminus region of human Trk C gp140. The NT-4/5 antibody (sc-545) was directed against an internal domain of human NT-4/5. The NGF antibody (sc-548) reacted to an amino terminus region of the mature form of human NGF. NT-3 antibody (sc-547) was directed against a carboxy terminus region of mature human NT-3. Anti-BDNF and anti-Trk B antibodies were used at 1:500 dilution for immunocytochemistry. All other antibodies were used at 1:1000 dilution for staining. Blocking peptides were used at 20:1 (peptide:antibody, micrograms) for primary antibody neutralization studies.
1131 Cells or oocytes were incubated 1 hour at room temperature in biotinylated second antibody (goat antirabbit IgG, 1:200) and then for 60 minutes in avidinbiotinylated enzyme complex (ABC reagent, Vectastain Elite, Vector Laboratories). Between each treatment, cells or oocytes were rinsed 3 times in PBS. The final buffer was replaced with diaminobenzidine tetrahydrochloride (DAB) under stereoscopic vision, with stain intensity recorded 10 minutes after addition of DAB. For a negative control, cells or oocytes were treated as described previously, except that the primary antibody was omitted. To demonstrate specificity of each receptor antibody, primary antibodies were preadsorbed with the peptide mimicking the receptor amino acid sequence against which the antibody was directed. Neurotrophin antisera were preadsorbed with intact neurotrophin protein. Both negative control and blocking controls were included in each staining process of cells and oocytes. Positive controls included staining of rat superior cervical ganglion cells for Trk receptors and their respective neurotrophin ligands.
Results Figure 1 shows positive immunocytochemistry staining of cumulus granulosa cells for each of the four neurotrophins (NT-4/5, BDNF, NT-3, NGF). Figure 2 shows immunocytochemistry staining of cumulus and unfertilized oocytes (day 0-day 2) for each their 3 respective Trk receptors (Trk A, Trk B, Trk C). Not shown here, is similar positive staining for mural granulosa for each of the neurotrophins and their respective receptors. BDNF and NT-4/5 staining intensity was similar in both cytoplasm and nucleus of the cumulus and mural cells but NT-3 and NGF appeared to be more localized in the nuclei of both cumulus and mural cells. There was no unequivocal difference in staining patterns between mural and cumulus granulosa cells. There was no difference in staining between day 0, 1, and 2 oocytes. Cells or oocytes not exposed to primary antibody (negative controls) and cells or oocytes exposed to preadsorbed antisera for each neurotrophin or Trk receptor (blocking controls) demonstrated significantly less intense or absent peroxidase reaction (Figures 1 and 2).
Comment These data demonstrate for the first time that neurotrophins and their respective receptor proteins are present in specific cell types within preovulatory ovarian follicles from women undergoing follicular aspiration for IVF/ICSI. More specifically, they demonstrate the presence of each of the 4 neurotrophins and their 3 respective Trk receptors within the mural and cumulus granulosa cells in addition to the presence of their 3 Trk receptors in the human oocyte. These observations
1132
Seifer, Feng, and Shelden
Figure 1 Immunocytochemistry staining of neurotrophins in human cumulus cells. A, Human cumulus cells stained without primary antibody for NT-4/5 (negative control). B, Human cumulus cells stained for NT-4/5. C, Human cumulus cells stained for NT4/5 using the NT-4/5 antibody that has been pre-adsorbed with NT-4/5 blocking peptide. D, E, F, show positive staining of cumulus for NGF, BDNF and NT-3 respectively. The scale bar represents 100 mm. Magnification of cumulus cells (!300).
support our previous conclusions17,18 that neurotrophins BDNF, NT-4/5, and NT-3 are produced within the follicle (specifically by granulosa) and provide evidence for the first time that the human preovulatory follicle also produces NGF. These data also demonstrate for the first time the presence of the Trk B, Trk C, and Trk A receptor proteins in the human oocyte and that these receptors are present before and after germinal vesicle breakdown. The location of the neurotrophins and their receptors suggest both an autocrine (granulosa) and a paracrine (granulosa-oocyte and granulosa-granulosa) function. Based on our previous work,17,18 we expected the present study would demonstrate that Trk B but not the Trk C receptor would be localized in the human oocyte. However, this was not the case, as each of the three Trk receptors (B, C, A) was demonstrated in the human preovulatory oocyte before and after germinal vesicle breakdown. We suspect that this difference in the presence of the Trk C receptor may represent species-specific differences between mouse and human oocytes. Most recently a study examining human ovarian sections from women undergoing surgery demonstrated immunocytochemical and reverse transcriptase polymerase chain reaction (RT-PCR) evidence for the presence of Trk A in oocytes and NGF in granulosa and oocytes in preantral follicles.22 Like NT-4/5 and NT-3, BDNF had previously been demonstrated in the follicular fluid of women
undergoing oocyte aspiration in preparation for IVF.17,18 With the use of ELISAs in conjunction with mural and cumulus granulosa cell culture, the previous studies demonstrated obvious BDNF protein secretion from cumulus but not mural granulosa cells. Our present findings of immunocytochemical staining of both the cumulus and granulosa cell nucleus and cytoplasm for each of the neurotrophins may support the concept of neurotrophin production in both types of granulosa cells but with neurotrophin secretion predominantly from cumulus granulosa cells. The relevance of a difference in intensity of staining between the cytoplasm and the nucleus of granulosa for specific neurotrophins will require further study. Previous work by Anderson et al23 in the fetal human ovary is consistent with our previous findings regarding Trk B in the adult ovary.17 They demonstrated the expression of messenger RNA (mRNA) for NT-4/5 and Trk B by using RT-PCR. In addition, Anderson et al showed NT-4/5 mRNA expression using in situ hybridization and NT-4/5 and Trk B using immunohistochemistry in the midtrimester fetal human ovary (13-21 weeks’ gestation). Specifically, NT-4/5 is present within fetal granulosa cells while Trk B is noted in the fetal oocyte thus suggesting that neurotrophins play a role in human follicular development and function. This role was further characterized in a follow-up study by the same group of investigators24 who demonstrated the important role Trk receptors play in the initial
Seifer, Feng, and Shelden
1133
Figure 2 Immunocytochemistry staining of neurotrophin receptors in human cumulus cells and unfertilized oocytes. A, B, C show positive staining of human cumulus cells for Trk A, Trk B, and Trk C, respectively. E, F, G show positive staining of human unfertilized oocytes for neurotrophin receptor Trk A, Trk B, and Trk C, respectively, with representive blocking control in D. The scale bar represents 100 mm. Magnification of oocytes (!120).
follicle formation within the human fetal ovary. Human fetal ovaries (13-16 weeks’ gestation) that were cultured in K252a, a potent inhibitor of Trk receptors, demonstrated a marked reduction in oogonia formation. Thus, it appears that ovarian neurotrophins and their receptors are present in the human fetus and are conserved in the adult. Although the presence of neurotrophins and their receptors in the human ovary is now evident, their physiologic roles are only beginning to be defined. In the mouse, Kawamura et al25 have most recently corroborated our findings regarding the role of BDNF in promoting first polar body extrusion of oocytes via the presence of the Trk B receptor. They further demonstrated the role of BDNF in promoting the development of mouse zygotes to blastocysts. Their findings support the concept that ovarian BDNF is vital to nuclear and cytoplasmic maturation of the mouse oocyte allowing it to progress to the blastocyst stage. Although a similar role in the human oocyte remains to be demonstrated, it is understood that the actions of any given ovarian growth factor may be cell site specific as well as temporally dependent on the presence or absence of other growth factor families. Neurotrophins like most other ovarian growth factors have been identified individually and will require additional investigation to better define their precise role in the overall context of an
intrafollicular oocyte-somatic cell communication network that is responsible for normal folliculogenesis. In another recent bovine study, Martins da Silva et al26 have further demonstrated that cellular localization and perhaps temporal activity of neurotrophins and their receptors may be species dependent. These investigators were unable to identify either mRNA transcripts or Trk B protein in bovine oocytes, in contrast to findings in the mouse and human. At the same time, BDNF mRNA transcripts and BDNF protein were identified in bovine cumulus cells. These investigators concluded from this work that BDNF may be involved in the cytoplasmic events of bovine oocyte maturation but that BDNF involvement in nuclear maturation (germinal vesicle breakdown to first polar body extrusion) was unlikely. Meanwhile, our recent work has demonstrated the presence of BDNF in preovulatory follicles of normally cycling women.19 We also have shown that BDNF production by granulosa cells in vitro can be stimulated by LH/hCG.20 These serial observations17-20 suggest that some neurotrophins, if not obligatory, may facilitate regulation of oocyte physiology, including oocyte development within preovulatory follicles. The current findings may encourage further investigation into the role(s) served by members of the neurotrophin family in oocyte maturation and eventually lead to new opportunities for treatment of infertility and/or methods of contraception.
1134
Seifer, Feng, and Shelden
References 1. Braude P, Bolton V, Moore S. Human gene expression first occurs between the four- and eight-cell stages of preimplantation development. Nature 1988;332:459-61. 2. Fair T. Follicular oocyte growth and acquisition of developmental competence. Anim Reprod Sci 2003;78:203-16. 3. Eppig JJ, Wigglesworth K, Pendola FL. The mammalian oocyte orchestrates the rate of ovarian follicular development. Proc Natl Acad Sci U S A 2002;99:2890-4. 4. Eppig JJ. Oocyte control of ovarian follicular development and function in mammals. Reproduction 2001;122:829-38. 5. Su YQ, Wigglesworth K, Pendola FL, O’Brien MJ, Eppig JJ. Mitogen-activated protein kinase activity in cumulus cells is essential for gonadotropin-induced oocyte meiotic resumption and cumulus expansion in the mouse. Endocrinology 2002;143:2221-32. 6. Shimasaki S, Moore RK, Otsuka F, Erickson GF. The bone morphogenetic protein system in mammalian reproduction. Endocrine Rev 2004;25:72-101. 7. Simon AM, Goodenough DA. Diverse functions of vertebrate gap junctions. Cell Biology 1998;8:477-83. 8. Tessarollo L. Pleiotropic functions of neurotrophins in development. Cytokine Growth Factor Rev 1998;9:125-37. 9. Yamamoto M, Sobue G, Yamamoto K, Terao S, Mitsuma T. Expression of mRNAs for neurotrophic factors (NGF, BDNF, NT-3 and GDNF) and their receptors (p75NGFR, Trk A, Trk B, and Trk C) in the adult human peripheral nervous system and nonneural tissues. Neurochem Res 1996;21:929-38. 10. Dissen GA, Hill DF, Costa ME, Dees WL, Lara HE, Ojeda SR. A role for Trk A nerve growth receptors in mammalian ovulation. Endocrinology 1996;137:198-209. 11. Dissen GA, Mayerhofer A, Ojeda SR. Participation of nerve growth factor in the regulation of ovarian function. Zygote 1996;4:309-12. 12. Mayerhofer A, Dissen GA, Parrott JA, Hill DF, Mayerhofer D, Garfield RE, et al. Involvement of nerve growth factor in the ovulatory cascade: trk A receptor activation inhibits gap junctional communication between thecal cells. Endocrinology 1996;137:5662-70. 13. Waraksa JA, Lindsay RM, Ip NY, Hutz RJ. Neurotrophin-3 augments steroid secretion by hamster ovarian follicles in vitro. Zoolog Sci 1995;12:499-502. 14. Dissen GA, Hirshfield AN, Malamed S, Ojeda SR. Expression of neurotrophins and their receptors in the mammalian ovary is developmentally regulated: changes at the time of folliculogenesis. Endocrinology 1995;136:4681-92. 15. Ojeda SR, Romero C, Tapia V, Dissen GA. Neurotrophic and cell-cell dependent control of early follicular development. Mol Cell Endocrinol 2000;163:67-71. 16. Paredes A, Romero C, Dissen GA, DeChiara TM, Reichardt L, Cornea A, et al. TrkB receptors are required for follicular growth and oocyte survival in the mammalian ovary. Dev Biol 2004;270: 430-49. 17. Seifer DB, Feng B, Shelden RM, Chen S, Dreyfus CF. Brainderived neurotrophic factor: a novel human ovarian follicular protein. J Clin Endocrinol Metab 2002;87:655-9. 18. Seifer DB, Feng B, Shelden RM, Chen S, Dreyfus CF. Neurotrophin-4/5 and Neurotrophin-3 are present within the human ovarian follicle but appear to have different paracrine/autocrine functions. J Clin Endocrinol Metab 2002;87:4569-71. 19. Seifer DB, Lambert-Messerlian G, Schneyer AL. Ovarian brainderived neurotrophic factor (BDNF) is present in follicular fluid from normally cycling women. Fertil Steril 2003;79:451-2. 20. Feng B, Chen S, Shelden RM, Seifer DB. Effect of gonadotropins on brain derived neurotrophic factor secretion by human follicular cumulus cells. Fertil Steril 2003;80:658-9. 21. Seifer DB, Gardiner AC, Lambert-Messerlian G, Schneyer AL. Differential secretion of dimeric inhibin in cultured luteinized
22.
23.
24.
25.
26.
granulosa cells as a function of ovarian reserve. J Clin Endocrinol Metab 1996;81:736-9. Abir R, Fisch B, Jin S, Ben-Haroush A, Felz C, Kessler-Icekson G, et al. Presence of NGF and its receptors in ovaries from human fetuses and adults. Mol Hum Reprod 2005;11:229-36. Anderson RA, Robinson LL, Brooks J, Spears N. Neurotropins and their receptors are expressed in the human fetal ovary. J Clin Endocrinol Metab 2002;87:890-7. Spears N, Molinek MD, Robinson LL, Fulton N, Cameron H, Shimoda K, et al. The role of neurotrophin receptors in female germcell survival in mouse and human. Development 2003;130:5481-91. Kawamura K, Kawamura K, Mulders SM, Gelpke MDS, Hsueh AJW. Ovarian brain-derived neurotrophic factor (BDNF) promotes the development of oocytes into preimplantation embryos. PNAS 2005;102:9206-11. Martins da Silva SJ, Gardner JO, Taylor JE, Springbett A, De Sousa PA, Anderson RA. Brain-derived neurotrophic factor promotes bovine oocyte cytoplasmic competence for embryo development. Reproduction 2005;129:423-34.
Discussion CHRISTOS COUTIFARIS, MD, PhD. I first want to thank the Society for asking me to discuss this paper on this group of factors and their receptors, which may very well prove to have a very important role in follicular development and the control of a normal reproductive lifespan. It is well known that the total number of primordial follicles in the human ovary reaches approximately 6 million while the female fetus is still in utero at mid gestation. From that point on, there is a progressive decline, which results in approximately 1 million being present at birth, 300,000 at the onset of puberty and reaching ‘‘0’’ at the time of menopause. A quick calculation suggests that only 300 to 600 of these follicles will result in ovulation of a mature oocyte during a woman’s reproductive lifespan. The only well-accepted fact about this process is that for this physiologic rate of decline in the follicular pool to occur, 2 intact X chromosomes are needed. Conditions in which all or part of an X chromosome is missing, such as in Turner syndrome, Turner mosaics, or cases of interstitial deletions of the long arm of the X, this process of follicular attrition is accelerated leading to premature ovarian failure. Starting in utero, primordial follicles, which contain oocytes arrested in prophase of the first meiotic division, are recruited continuously to enter the pool of developing follicles, some of which (during the reproductive years) become dominant leading to the ovulation of a mature oocyte, or become atretic and the containing oocytes are permanently lost from the total gamete pool. Although this ‘‘dogma’’ has been recently challenged with the suggestion of the existence of stem cells capable of producing new oocytes, these novel findings remain to be confirmed. The mechanisms that regulate this primordial to primary follicle transition, and what is the ‘‘molecular
www.ajog.org
Immunocytochemical evidence for the presence and location of the neurotrophin–Trk receptor family in adult human preovulatory ovarian follicles David B. Seifer, MD,* Bo Feng, PhD, Robert M. Shelden, PhD Department of Obstetrics, Gynecology and Reproductive Sciences, Division of Reproductive Endocrinology and Infertility, UMDNJ-Robert Wood Johnson Medical School, New Brunswick, NJ Received for publication November 14, 2005; accepted December 15, 2005
KEY WORDS Neurotrophins Trk receptors Granulosa Oocytes Follicles Growth factors Ovary
Objective: This study was undertaken to evaluate the presence or absence of neurotrophins and their respective receptors within adult human preovulatory follicles. Study design: Prospective study of neurotrophins and their receptors in follicular cells and unfertilized oocytes from women undergoing aspiration for in vitro fertilization/intracytoplasmic sperm injection. Cells (mural and cumulus granulosa cells, unfertilized oocytes) were examined for immunocytochemical staining of neurotrophin and receptor proteins. Results: Mural and cumulus granulosa cells were positive for BDNF, NT-4/5, NT-3, and NGF, as well as for Trk B, Trk C, and Trk A receptors. Unfertilized oocytes were positive for Trk B, Trk C, and Trk A receptors. Conclusion: Neurotrophins and their respective receptor proteins are present within the mural and cumulus granulosa cells of adult human preovulatory follicles. Neurotrophin receptors are present in human unfertilized oocytes. The location of the neurotrophins and their receptors suggest both an autocrine and paracrine function within the adult human ovarian follicle. Ó 2006 Mosby, Inc. All rights reserved.
Development and maturation of vertebrate gametes is the culmination of a highly complex series of processes involving nonovarian (eg, hypothalamic releasing/stimulating factors, hypophyseal gonadotropin hormones), ovarian (steroid hormones), and cellular (cell signaling)
Supported in part by the Samuel A. Pasquale grant. Presented at the Twenty-Fourth Annual Meeting of the American Gynecological and Obstetrical Society, September 29-October 1, 2005, Victoria, British Columbia, Canada. * Reprint requests: David B. Seifer, MD, Maimonides Medical Center, 1355 84th St, Brooklyn, NY 11228. E-mail: [email protected] 0002-9378/$ - see front matter Ó 2006 Mosby, Inc. All rights reserved. doi:10.1016/j.ajog.2005.12.022
inputs exquisitely orchestrated to produce gametes capable of fertilization. The oocyte carries the additional requirement for storing sufficient information and resources to support the newly formed zygote through 2 or more cell divisions until the newly emerging embryo genome is able to assume command of subsequent development.1,2 Although much remains to be elucidated, endocrine regulation of the hypothalamic-hypophyseal-gonadal axis through positive and negative feedback loops has been well established. Intragonadal and intracellular regulatory pathways and mechanisms are less well appreciated, but significant gains in understanding of
1130 these processes have occurred. It is, for example, well established through the work of Eppig and others3-5 that the oocyte not only receives information and material from the surrounding granulosa cells (corona radiata), but also plays a highly significant reciprocal role in regulating the activity and production of these cells. Moreover, it is clear that a multitude of growth factors, eg, growth and development factor-9 (GDF-9), insulinlike growth factor (IGF), transforming growth factor (TGF-ß), bone morphogenetic protein (BMP)6 and adhesion factors, eg, connexins,7 are critical in the developmental processes involved in the intrafollicular oocyte-somatic cell communication network that leads to mature oocytes. More recently, it has been recognized that neurotrophins, a family of soluble polypeptide growth factors well known for their role in neuronal survival and neural outgrowth, may be intimately involved in the dynamic development and maturation of the ovarian follicle/ oocyte complex. Although other neurotrophic factors have been identified, the neurotrophins found in a variety of non-neuronal systems (eg, cardiovascular, immune, endocrine, and reproductive systems8,9) include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-4/5 (NT-4/5), and neurotrophin-3 (NT-3). BDNF, NT-4/5, NT-3, and NGF and their respective tyrosine kinase (Trk) receptors (Trk B for BDNF and NT-4/5, Trk C for NT-3, and Trk A for NGF) have been identified in the mammalian ovary10 and have been shown to play a role in ovulation,11,12 steroid secretion,13 and follicular development14-16 in the rodent. Recently, we described presumptive evidence for the secretion of neurotrophins and the presence of their receptors in human cumulus cells.17-20 We first reported the presence of BDNF in the follicular fluid of the human ovarian follicle. We demonstrated its secretion by human cumulus granulosa cells and its promotion of mouse oocyte maturation presumably via its preferred receptor, Trk B.17 We reported the presence of NT-4/5, also exhibiting preferential binding to the Trk B receptor, and NT-3, a non-Trk B ligand within the human ovarian follicle.18 We further demonstrated the presence of Trk B receptor in the immature mouse oocyte and BDNF-induced acceleration of first polar body formation by cultured immature mouse oocytes.17,18 It is emphasized that these prior studies examined the majority of neurotrophins secreted in specifically follicular fluid and cell culture media, not directly by cells per se, using commercially available enzyme-linked immunosorbent assays (ELISAs) with investigation of neurotrophin receptor presence limited to only the examination of mouse oocytes.17,18 In the current study, we extended these investigations to include direct immunocytochemical evidence of neurotrophins and their respective receptors in the basic
Seifer, Feng, and Shelden cellular components of the human follicle that include mural granulosa cells, cumulus granulosa cells, and oocytes obtained from women undergoing oocyte aspiration for in vitro fertilization. Our objective was to determine whether neurotrophins and their respective receptors are present within the adult human preovulatory follicles of women undergoing follicular aspiration for in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI).
Material and methods Population Follicular fluid was obtained from 29 women younger than 42 years undergoing follicular aspiration for IVF/ ICSI. Women had received either leuprolide acetate (Lupron, TAP Pharmaceuticals, Inc, North Chicago, IL) for pituitary desensitization, followed by 2 to 8 ampules of gonadotropins given daily in divided morning and evening doses as previously described21 or daily gonadotropins until a lead follicle was 14 mm or greater in mean diameter whereupon cetrorelix acetate (Cetrotide, Serono, Norwell, MA) was added to prevent premature ovulation. Transvaginal follicular aspiration was performed under sedation 36 hours after administration of 5,000 or 10,000 IU hCG when at least 4 lead follicles were 18 mm or greater in mean diameter. The UMDNJRobert Wood Johnson Medical School Institutional Review Board approved this study protocol.
Human mural and cumulus granulosa cell culture Pooled clear follicular fluid was centrifuged 10 minutes, 325!g at 20(C. The top layer of cells enriched with mural granulosa was collected into Hanks’ Balanced Salt Solution (HBSS, Ca and Mg-free, Sigma, St Louis, MO) then overlaid on top of 45% colloidal silica solution (Enhance-S Plus, Conception Technologies, San Diego, CA) and centrifuged for 20 minutes at 325!g. After separation, the mural granulosa cell enriched interface layer was collected and washed in HBSS. The resultant mural granulosa cell pellet was incubated in collagenase type IA (100 m/mL, Sigma) at room temperature until cell clumps separated. Cells were washed twice then plated in 8-chamber slides (Nalge Nunc International Corp, Naperville, IL). Cumulus granulosa cells were removed from oocytes in preparation for IVF/ICSI and then were incubated with hyaluronidase (100 IU/mL, Sigma), followed by 2! wash with HBSS. For each experiment, cumulus cells from 2 to 3 patients were pooled, then plated onto 8-chamber slides. Both cell types (mural and cumulus granulosa) were cultured in Ham’s F-10 medium supplemented with 10% fetal bovine serum (FBS) at 37(C, 5% CO2 in air. Each experiment was carried out a minimum of 3 times.
Seifer, Feng, and Shelden
Human oocytes Ninety-eight human unfertilized oocytes were obtained day 0 to day 2 from 31 consenting patients undergoing IVF/ICSI after retrieval on day 0. Day 0 oocytes were immature (germinal vesicle or Meiosis I stage), whereas day 1 and day 2 oocytes were mature (first polar body present) on day 0 but did not exhibit pronuclei after insemination or injection. Isolated oocytes were washed twice with phosphate-buffered saline solution (PBS) and were fixed with 4% paraformaldehyde for 10 minutes, followed by PBS wash. Fixed oocytes were stored at 4(C in blocking buffer (1.5% normal goat serum in PBS) until used for immunocytochemistry procedures described below. Oocyte (day 0-2) Trk staining was repeated in at least 3 independent staining procedures. Photographs were taken immediately after the final wash using an Imagine Photographer (Scanalytics, Inc, Fairfax, VA).
Immunocytochemistry staining After an initial 24-hour culture, granulosa cells were washed twice with PBS then fixed with 4% paraformaldehyde for 10 minutes, followed by 3! wash with PBS. Immunocytochemistry staining of neurotrophins and their receptors was carried out at room temperature using ABC reagent (Vectastain Elite, Vector Laboratories, Burlingame, CA). In brief, fixed cells or oocytes were treated with blocking serum (1.5% goat serum with 0.3% Triton-X100 in PBS). After washing with PBS, cells or oocytes were incubated overnight at 4(C with primary antibody against BDNF (Promega, Madison, WI), NT-3, NT-4/5, NGF, Trk A, Trk B, Trk C (Santa Cruz Biotechnology, Inc, Santa Cruz, CA). Antibodies used in these studies were affinity purified polyclonal rabbit antibodies that demonstrated no cross-reactivity with related Trk or neurotrophin species. Trk A antibody (sc-118) was directed against a carboxy terminus region of the gp140 precursor form of human Trk A. Trk B antibody (sc-12) was directed against a carboxy terminus region of the human Trk B precursor gp145. The Trk B antibody binds to Trk B of mouse, rat, and human origin but does not bind to the truncated form of Trk B. Trk C (sc-117) antibody was directed against a carboxy terminus region of human Trk C gp140. The NT-4/5 antibody (sc-545) was directed against an internal domain of human NT-4/5. The NGF antibody (sc-548) reacted to an amino terminus region of the mature form of human NGF. NT-3 antibody (sc-547) was directed against a carboxy terminus region of mature human NT-3. Anti-BDNF and anti-Trk B antibodies were used at 1:500 dilution for immunocytochemistry. All other antibodies were used at 1:1000 dilution for staining. Blocking peptides were used at 20:1 (peptide:antibody, micrograms) for primary antibody neutralization studies.
1131 Cells or oocytes were incubated 1 hour at room temperature in biotinylated second antibody (goat antirabbit IgG, 1:200) and then for 60 minutes in avidinbiotinylated enzyme complex (ABC reagent, Vectastain Elite, Vector Laboratories). Between each treatment, cells or oocytes were rinsed 3 times in PBS. The final buffer was replaced with diaminobenzidine tetrahydrochloride (DAB) under stereoscopic vision, with stain intensity recorded 10 minutes after addition of DAB. For a negative control, cells or oocytes were treated as described previously, except that the primary antibody was omitted. To demonstrate specificity of each receptor antibody, primary antibodies were preadsorbed with the peptide mimicking the receptor amino acid sequence against which the antibody was directed. Neurotrophin antisera were preadsorbed with intact neurotrophin protein. Both negative control and blocking controls were included in each staining process of cells and oocytes. Positive controls included staining of rat superior cervical ganglion cells for Trk receptors and their respective neurotrophin ligands.
Results Figure 1 shows positive immunocytochemistry staining of cumulus granulosa cells for each of the four neurotrophins (NT-4/5, BDNF, NT-3, NGF). Figure 2 shows immunocytochemistry staining of cumulus and unfertilized oocytes (day 0-day 2) for each their 3 respective Trk receptors (Trk A, Trk B, Trk C). Not shown here, is similar positive staining for mural granulosa for each of the neurotrophins and their respective receptors. BDNF and NT-4/5 staining intensity was similar in both cytoplasm and nucleus of the cumulus and mural cells but NT-3 and NGF appeared to be more localized in the nuclei of both cumulus and mural cells. There was no unequivocal difference in staining patterns between mural and cumulus granulosa cells. There was no difference in staining between day 0, 1, and 2 oocytes. Cells or oocytes not exposed to primary antibody (negative controls) and cells or oocytes exposed to preadsorbed antisera for each neurotrophin or Trk receptor (blocking controls) demonstrated significantly less intense or absent peroxidase reaction (Figures 1 and 2).
Comment These data demonstrate for the first time that neurotrophins and their respective receptor proteins are present in specific cell types within preovulatory ovarian follicles from women undergoing follicular aspiration for IVF/ICSI. More specifically, they demonstrate the presence of each of the 4 neurotrophins and their 3 respective Trk receptors within the mural and cumulus granulosa cells in addition to the presence of their 3 Trk receptors in the human oocyte. These observations
1132
Seifer, Feng, and Shelden
Figure 1 Immunocytochemistry staining of neurotrophins in human cumulus cells. A, Human cumulus cells stained without primary antibody for NT-4/5 (negative control). B, Human cumulus cells stained for NT-4/5. C, Human cumulus cells stained for NT4/5 using the NT-4/5 antibody that has been pre-adsorbed with NT-4/5 blocking peptide. D, E, F, show positive staining of cumulus for NGF, BDNF and NT-3 respectively. The scale bar represents 100 mm. Magnification of cumulus cells (!300).
support our previous conclusions17,18 that neurotrophins BDNF, NT-4/5, and NT-3 are produced within the follicle (specifically by granulosa) and provide evidence for the first time that the human preovulatory follicle also produces NGF. These data also demonstrate for the first time the presence of the Trk B, Trk C, and Trk A receptor proteins in the human oocyte and that these receptors are present before and after germinal vesicle breakdown. The location of the neurotrophins and their receptors suggest both an autocrine (granulosa) and a paracrine (granulosa-oocyte and granulosa-granulosa) function. Based on our previous work,17,18 we expected the present study would demonstrate that Trk B but not the Trk C receptor would be localized in the human oocyte. However, this was not the case, as each of the three Trk receptors (B, C, A) was demonstrated in the human preovulatory oocyte before and after germinal vesicle breakdown. We suspect that this difference in the presence of the Trk C receptor may represent species-specific differences between mouse and human oocytes. Most recently a study examining human ovarian sections from women undergoing surgery demonstrated immunocytochemical and reverse transcriptase polymerase chain reaction (RT-PCR) evidence for the presence of Trk A in oocytes and NGF in granulosa and oocytes in preantral follicles.22 Like NT-4/5 and NT-3, BDNF had previously been demonstrated in the follicular fluid of women
undergoing oocyte aspiration in preparation for IVF.17,18 With the use of ELISAs in conjunction with mural and cumulus granulosa cell culture, the previous studies demonstrated obvious BDNF protein secretion from cumulus but not mural granulosa cells. Our present findings of immunocytochemical staining of both the cumulus and granulosa cell nucleus and cytoplasm for each of the neurotrophins may support the concept of neurotrophin production in both types of granulosa cells but with neurotrophin secretion predominantly from cumulus granulosa cells. The relevance of a difference in intensity of staining between the cytoplasm and the nucleus of granulosa for specific neurotrophins will require further study. Previous work by Anderson et al23 in the fetal human ovary is consistent with our previous findings regarding Trk B in the adult ovary.17 They demonstrated the expression of messenger RNA (mRNA) for NT-4/5 and Trk B by using RT-PCR. In addition, Anderson et al showed NT-4/5 mRNA expression using in situ hybridization and NT-4/5 and Trk B using immunohistochemistry in the midtrimester fetal human ovary (13-21 weeks’ gestation). Specifically, NT-4/5 is present within fetal granulosa cells while Trk B is noted in the fetal oocyte thus suggesting that neurotrophins play a role in human follicular development and function. This role was further characterized in a follow-up study by the same group of investigators24 who demonstrated the important role Trk receptors play in the initial
Seifer, Feng, and Shelden
1133
Figure 2 Immunocytochemistry staining of neurotrophin receptors in human cumulus cells and unfertilized oocytes. A, B, C show positive staining of human cumulus cells for Trk A, Trk B, and Trk C, respectively. E, F, G show positive staining of human unfertilized oocytes for neurotrophin receptor Trk A, Trk B, and Trk C, respectively, with representive blocking control in D. The scale bar represents 100 mm. Magnification of oocytes (!120).
follicle formation within the human fetal ovary. Human fetal ovaries (13-16 weeks’ gestation) that were cultured in K252a, a potent inhibitor of Trk receptors, demonstrated a marked reduction in oogonia formation. Thus, it appears that ovarian neurotrophins and their receptors are present in the human fetus and are conserved in the adult. Although the presence of neurotrophins and their receptors in the human ovary is now evident, their physiologic roles are only beginning to be defined. In the mouse, Kawamura et al25 have most recently corroborated our findings regarding the role of BDNF in promoting first polar body extrusion of oocytes via the presence of the Trk B receptor. They further demonstrated the role of BDNF in promoting the development of mouse zygotes to blastocysts. Their findings support the concept that ovarian BDNF is vital to nuclear and cytoplasmic maturation of the mouse oocyte allowing it to progress to the blastocyst stage. Although a similar role in the human oocyte remains to be demonstrated, it is understood that the actions of any given ovarian growth factor may be cell site specific as well as temporally dependent on the presence or absence of other growth factor families. Neurotrophins like most other ovarian growth factors have been identified individually and will require additional investigation to better define their precise role in the overall context of an
intrafollicular oocyte-somatic cell communication network that is responsible for normal folliculogenesis. In another recent bovine study, Martins da Silva et al26 have further demonstrated that cellular localization and perhaps temporal activity of neurotrophins and their receptors may be species dependent. These investigators were unable to identify either mRNA transcripts or Trk B protein in bovine oocytes, in contrast to findings in the mouse and human. At the same time, BDNF mRNA transcripts and BDNF protein were identified in bovine cumulus cells. These investigators concluded from this work that BDNF may be involved in the cytoplasmic events of bovine oocyte maturation but that BDNF involvement in nuclear maturation (germinal vesicle breakdown to first polar body extrusion) was unlikely. Meanwhile, our recent work has demonstrated the presence of BDNF in preovulatory follicles of normally cycling women.19 We also have shown that BDNF production by granulosa cells in vitro can be stimulated by LH/hCG.20 These serial observations17-20 suggest that some neurotrophins, if not obligatory, may facilitate regulation of oocyte physiology, including oocyte development within preovulatory follicles. The current findings may encourage further investigation into the role(s) served by members of the neurotrophin family in oocyte maturation and eventually lead to new opportunities for treatment of infertility and/or methods of contraception.
1134
Seifer, Feng, and Shelden
References 1. Braude P, Bolton V, Moore S. Human gene expression first occurs between the four- and eight-cell stages of preimplantation development. Nature 1988;332:459-61. 2. Fair T. Follicular oocyte growth and acquisition of developmental competence. Anim Reprod Sci 2003;78:203-16. 3. Eppig JJ, Wigglesworth K, Pendola FL. The mammalian oocyte orchestrates the rate of ovarian follicular development. Proc Natl Acad Sci U S A 2002;99:2890-4. 4. Eppig JJ. Oocyte control of ovarian follicular development and function in mammals. Reproduction 2001;122:829-38. 5. Su YQ, Wigglesworth K, Pendola FL, O’Brien MJ, Eppig JJ. Mitogen-activated protein kinase activity in cumulus cells is essential for gonadotropin-induced oocyte meiotic resumption and cumulus expansion in the mouse. Endocrinology 2002;143:2221-32. 6. Shimasaki S, Moore RK, Otsuka F, Erickson GF. The bone morphogenetic protein system in mammalian reproduction. Endocrine Rev 2004;25:72-101. 7. Simon AM, Goodenough DA. Diverse functions of vertebrate gap junctions. Cell Biology 1998;8:477-83. 8. Tessarollo L. Pleiotropic functions of neurotrophins in development. Cytokine Growth Factor Rev 1998;9:125-37. 9. Yamamoto M, Sobue G, Yamamoto K, Terao S, Mitsuma T. Expression of mRNAs for neurotrophic factors (NGF, BDNF, NT-3 and GDNF) and their receptors (p75NGFR, Trk A, Trk B, and Trk C) in the adult human peripheral nervous system and nonneural tissues. Neurochem Res 1996;21:929-38. 10. Dissen GA, Hill DF, Costa ME, Dees WL, Lara HE, Ojeda SR. A role for Trk A nerve growth receptors in mammalian ovulation. Endocrinology 1996;137:198-209. 11. Dissen GA, Mayerhofer A, Ojeda SR. Participation of nerve growth factor in the regulation of ovarian function. Zygote 1996;4:309-12. 12. Mayerhofer A, Dissen GA, Parrott JA, Hill DF, Mayerhofer D, Garfield RE, et al. Involvement of nerve growth factor in the ovulatory cascade: trk A receptor activation inhibits gap junctional communication between thecal cells. Endocrinology 1996;137:5662-70. 13. Waraksa JA, Lindsay RM, Ip NY, Hutz RJ. Neurotrophin-3 augments steroid secretion by hamster ovarian follicles in vitro. Zoolog Sci 1995;12:499-502. 14. Dissen GA, Hirshfield AN, Malamed S, Ojeda SR. Expression of neurotrophins and their receptors in the mammalian ovary is developmentally regulated: changes at the time of folliculogenesis. Endocrinology 1995;136:4681-92. 15. Ojeda SR, Romero C, Tapia V, Dissen GA. Neurotrophic and cell-cell dependent control of early follicular development. Mol Cell Endocrinol 2000;163:67-71. 16. Paredes A, Romero C, Dissen GA, DeChiara TM, Reichardt L, Cornea A, et al. TrkB receptors are required for follicular growth and oocyte survival in the mammalian ovary. Dev Biol 2004;270: 430-49. 17. Seifer DB, Feng B, Shelden RM, Chen S, Dreyfus CF. Brainderived neurotrophic factor: a novel human ovarian follicular protein. J Clin Endocrinol Metab 2002;87:655-9. 18. Seifer DB, Feng B, Shelden RM, Chen S, Dreyfus CF. Neurotrophin-4/5 and Neurotrophin-3 are present within the human ovarian follicle but appear to have different paracrine/autocrine functions. J Clin Endocrinol Metab 2002;87:4569-71. 19. Seifer DB, Lambert-Messerlian G, Schneyer AL. Ovarian brainderived neurotrophic factor (BDNF) is present in follicular fluid from normally cycling women. Fertil Steril 2003;79:451-2. 20. Feng B, Chen S, Shelden RM, Seifer DB. Effect of gonadotropins on brain derived neurotrophic factor secretion by human follicular cumulus cells. Fertil Steril 2003;80:658-9. 21. Seifer DB, Gardiner AC, Lambert-Messerlian G, Schneyer AL. Differential secretion of dimeric inhibin in cultured luteinized
22.
23.
24.
25.
26.
granulosa cells as a function of ovarian reserve. J Clin Endocrinol Metab 1996;81:736-9. Abir R, Fisch B, Jin S, Ben-Haroush A, Felz C, Kessler-Icekson G, et al. Presence of NGF and its receptors in ovaries from human fetuses and adults. Mol Hum Reprod 2005;11:229-36. Anderson RA, Robinson LL, Brooks J, Spears N. Neurotropins and their receptors are expressed in the human fetal ovary. J Clin Endocrinol Metab 2002;87:890-7. Spears N, Molinek MD, Robinson LL, Fulton N, Cameron H, Shimoda K, et al. The role of neurotrophin receptors in female germcell survival in mouse and human. Development 2003;130:5481-91. Kawamura K, Kawamura K, Mulders SM, Gelpke MDS, Hsueh AJW. Ovarian brain-derived neurotrophic factor (BDNF) promotes the development of oocytes into preimplantation embryos. PNAS 2005;102:9206-11. Martins da Silva SJ, Gardner JO, Taylor JE, Springbett A, De Sousa PA, Anderson RA. Brain-derived neurotrophic factor promotes bovine oocyte cytoplasmic competence for embryo development. Reproduction 2005;129:423-34.
Discussion CHRISTOS COUTIFARIS, MD, PhD. I first want to thank the Society for asking me to discuss this paper on this group of factors and their receptors, which may very well prove to have a very important role in follicular development and the control of a normal reproductive lifespan. It is well known that the total number of primordial follicles in the human ovary reaches approximately 6 million while the female fetus is still in utero at mid gestation. From that point on, there is a progressive decline, which results in approximately 1 million being present at birth, 300,000 at the onset of puberty and reaching ‘‘0’’ at the time of menopause. A quick calculation suggests that only 300 to 600 of these follicles will result in ovulation of a mature oocyte during a woman’s reproductive lifespan. The only well-accepted fact about this process is that for this physiologic rate of decline in the follicular pool to occur, 2 intact X chromosomes are needed. Conditions in which all or part of an X chromosome is missing, such as in Turner syndrome, Turner mosaics, or cases of interstitial deletions of the long arm of the X, this process of follicular attrition is accelerated leading to premature ovarian failure. Starting in utero, primordial follicles, which contain oocytes arrested in prophase of the first meiotic division, are recruited continuously to enter the pool of developing follicles, some of which (during the reproductive years) become dominant leading to the ovulation of a mature oocyte, or become atretic and the containing oocytes are permanently lost from the total gamete pool. Although this ‘‘dogma’’ has been recently challenged with the suggestion of the existence of stem cells capable of producing new oocytes, these novel findings remain to be confirmed. The mechanisms that regulate this primordial to primary follicle transition, and what is the ‘‘molecular