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WO1999050672A1 - Analyse pour facteur 9 de differenciation de croissance - Google Patents

Analyse pour facteur 9 de differenciation de croissance Download PDF

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Publication number
WO1999050672A1
WO1999050672A1 PCT/US1999/007210 US9907210W WO9950672A1 WO 1999050672 A1 WO1999050672 A1 WO 1999050672A1 US 9907210 W US9907210 W US 9907210W WO 9950672 A1 WO9950672 A1 WO 9950672A1
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WIPO (PCT)
Prior art keywords
gdf
binding
expression
gene
agent
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PCT/US1999/007210
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English (en)
Inventor
Martin M. Matzuk
Julia A. Elvin
Pei Wang
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Baylor College Of Medicine
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Publication date
Application filed by Baylor College Of Medicine filed Critical Baylor College Of Medicine
Priority to CA002325019A priority Critical patent/CA2325019A1/fr
Priority to NZ507484A priority patent/NZ507484A/xx
Priority to JP2000541529A priority patent/JP2002510055A/ja
Priority to MXPA00009653A priority patent/MXPA00009653A/es
Priority to AU33777/99A priority patent/AU753793B2/en
Priority to EP99915200A priority patent/EP1066528A1/fr
Publication of WO1999050672A1 publication Critical patent/WO1999050672A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/689—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to pregnancy or the gonads
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/475—Assays involving growth factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the oocyte plays an integral role in regulating folliculogenesis within the mammalian ovary.
  • the oocyte has been shown to act on granulosa cells to regulate follicle formation perinatally, stimulate granulosa cell proliferation, modulate granulosa cell gene expression, and influence steroidogenesis (Reviewed in (Eppig, J.J., Dev. BioL, 5:51-59 (1994)).
  • the granulosa cells in the pre-ovulatory follicle can be separated into two populations with regard to their proximity to the oocyte; cumulus granulosa cells closely surround the oocyte while the mural granulosa cells are located around the periphery of the follicle separated from the oocyte by an antrum. Cumulus cells secrete a hyaluronic acid-rich matrix during cumulus expansion and are extruded with the oocyte during ovulation.
  • This expanded matrix is a critical factor for reproductive integrity since it binds the oocyte and cumulus cells together, facilitates follicular extrusion and oviductal fimbria capture, and allows sperm penetration and fertilization (Salustri, A., et al., Zygote, 4:313-315 (1996)).
  • mural granulosa cells synthesize proteases important for follicle rupture at ovulation, remain within the ovary after the cumulus cell-oocyte complex is released, and eventually undergo terminal differentiation to form the corpus luteum.
  • Mouse growth differentiation factor 9 (mGDF-9) is expressed in the ovary and specifically in oocytes (PCT WO94/15966).
  • a GDF-9 knockout mouse demonstrated female infertility due to an early block in folliculogenesis (Dong et al, Nature, 355:531-535 (1996)), however, the GDF-9 receptor and the cell type expressing the GDF-9 receptor have not been identified. Discovery of the cell type expressing the GDF-9 receptor would assist in defining the role of GDF-9 in fertility, and would provide an assay system for identifying agents which target GDF-9 activity.
  • GDF-9 based upon the discovery that GDF-9 binds to granulosa cells found in the ovary of mammals. As also described herein, expression of particular proteins are enhanced and/or inhibited upon binding of GDF-9 to the receptors on the granulosa cells.
  • the present invention relates to a method of identifying an agent which alters
  • the method involves combining cells having receptors for GDF-9 (e.g., granulosa cells) and a gene (one or more) whose expression is regulated by binding of GDF-9 to the receptors (e.g., hyaluronan synthase, steroidogenic acute regulatory protein (StAR), luetinizng hormone (LH) receptor, cyclooxygenase 2 (COX-2), urokinase plasminogen activator (uPA), kit ligand, activin/inhibin ⁇ B and follistatin); GDF-9; and an agent to be assessed (test sample).
  • GDF-9 e.g., granulosa cells
  • a gene whose expression is regulated by binding of GDF-9 to the receptors (e.g., hyaluronan synthase, steroidogenic acute regulatory protein (StAR), luetinizng hormone (LH) receptor, cyclooxygenas
  • the combination produced in the test sample is maintained under conditions appropriate for binding of GDF-9 to the receptors on the cells.
  • the extent to which expression of the gene occurs is then determined, wherein alteration of expression of the gene upon binding of GDF-9 to the receptors in the presence of the agent to be assessed indicates that the agent alters GDF-9 activity.
  • the method can further comprise a control step wherein the extent to which binding occurs in the presence of the agent is compared to the extent to which binding occurs in the absence of the agent.
  • the extent to which expression of the gene occurs in the test sample is compared to the extent to which expression of the gene occurs in a control sample (e.g., a combination comprising the cells having receptors for GDF-9 and a gene whose expression is regulated by binding of GDF-9 to the receptors, and GDF-
  • the present invention also relates to a method of identifying an agent which is an inhibitor of GDF-9 activity.
  • granulosa cells; GDF-9; and an agent to be assessed are combined.
  • the combination produced is maintained under conditions appropriate for binding of GDF-9 to receptors on the granulosa cells; and the extent to which expression of a gene regulated by binding of GDF-9 to the receptors occurs upon binding of GDF-9 to the receptors on the granulosa cells is determined.
  • Inhibition of expression of the gene upon binding of GDF-9 to the receptors in the presence of the agent to be assessed indicates the agent inhibits GDF-9 activity.
  • granulosa cells; GDF-9; and an agent to be assessed are combined.
  • the combination produced is maintained under conditions appropriate for binding of GDF-9 to receptors on the granulosa cells; and the extent to which expression of uPA, a gene regulated by binding of GDF-9 to the receptors, occurs upon binding of GDF-9 to the receptors on the granulosa cells is determined. Increased expression of the uPA gene upon binding of GDF-9 to the receptors in the presence of the agent to be assessed indicates that the agent inhibits GDF-9 activity.
  • the present invention also relates to a method of identifying an agent which is an enhancer of GDF-9 activity. In one embodiment, granulosa cells; GDF-9; and an agent to be assessed are combined. The combination produced is maintained -4-
  • granulosa cells; GDF-9; and an agent to be assessed are combined.
  • the combination produced is maintained under conditions appropriate for binding of GDF-9 to receptors on the granulosa cells; and the extent to which expression of a uPA gene regulated by binding of GDF-9 to the receptors occurs upon binding of GDF-9 to the receptors on the granulosa cells is determined. Decreased expression of the uPA gene upon binding of GDF-9 to the receptors in the presence of the agent to be assessed indicates that the agent enhances GDF-9 activity.
  • the methods of the present invention can also be used to identify an agent which inhibits fertility in a mammal (e.g., human). Alternatively, the methods of the present invention can be used to identify an agent which enhances fertility in a mammal.
  • a mammal e.g., human
  • the methods of the present invention can be used to identify an agent which enhances fertility in a mammal.
  • the present invention also relates to a method of identifying an agent which is an agonist of GDF-9.
  • cells having receptors for GDF-9 and a gene, wherein expression of the gene is regulated by binding of GDF-9 to the receptor; GDF-9; and an agent to be assessed are combined.
  • the combination produced is maintained under conditions appropriate for binding of GDF-9 to receptors on the cells, and the extent to which expression of the gene regulated by binding of GDF-9 to the receptors occurs is determined, wherein expression of the gene in the presence of the agent to be assessed indicates that the agent is an agonist of GDF-9.
  • the present invention relates to a method of identifying an agent which is an agonist or antagonist of GDF-9.
  • cells having receptors for GDF-9 and a gene whose expression is regulated by binding of GDF-9 to the receptor; GDF-9 and an agent to be assessed are combined.
  • the combination produced is maintained under conditions appropriate for binding of the agent to receptors on the cells.
  • the extent to which binding of the agent to the receptors on the cells occurs is determined, wherein binding of the agent to the GDF-9 receptors indicates that the agent is an agonist or an antagonist of GDF-9.
  • the extent to which binding of GDF-9 to the receptor on the cells occurs can be determined in a variety of ways.
  • the extent to which binding occurs is determined by directly measuring a gene product (e.g., nucleic acids such as DNA, RNA of the gene; protein, peptide encoded by the gene) of the gene which is regulated by binding of GDF-9 to the receptors.
  • a gene product e.g., nucleic acids such as DNA, RNA of the gene; protein, peptide encoded by the gene
  • the gene encodes a protein involved in the synthesis of hyaluronic acid (e.g., hyaluronan synthase), and the extent to which binding of GDF-9 to the receptors on the granulosa cells occurs is determined by measuring the production of a product of the gene (e.g., RNA coding for hyaluronan synthase), wherein an increase in production the gene product indicates the agent is an enhancer of GDF-9 activity and a decrease of production of gene product indicates the agent is an inhibitor of GDF-9 activity.
  • a product of the gene e.g., RNA coding for hyaluronan synthase
  • the gene encodes a protein involved in the synthesis of progesterone (e.g., StAR), and the extent to which binding of GDF-9 to the receptors on the granulosa cells occurs is determined by measuring the production of a product of the gene (e.g., RNA coding for StAR), wherein an increase in the production of the gene product indicates that the agent is an enhancer of GDF-9 activity, and a decrease in production of the gene product indicates that the agent is an inhibitor of GDF-9 activity.
  • a product of the gene e.g., RNA coding for StAR
  • the gene encodes a protein involved in the production of plasmin (e.g., uPA), and the extent to which expression of the gene regulated by binding of GDF-9 to the receptors occurs upon binding of GDF-9 to the receptors is determined by measuring the production of a product of the gene (e.g., RNA coding for uPA), wherein a decrease in the expression of the gene product indicates the agent is an enhancer of GDF-9 activity, and an increase in expression of the gene product indicates the agent is an inhibitor of GDF-9 activity.
  • a product of the gene e.g., RNA coding for uPA
  • the extent to which binding of GDF-9 to the receptor on the cells occurs can also be determined by measuring a product or function (e.g., hyaluronic acid, progesterone and/or plasmin) attributed to the activity of proteins encoded by the gene whose expression is regulated by binding of GDF-9 to the receptor on the cell.
  • a product or function e.g., hyaluronic acid, progesterone and/or plasmin
  • granulosa cells respond to GDF-9 and that this response can be determined by means of one or more inducible genes allows for an in vitro bioassay for GDF-9.
  • Such an assay can be used to diagnose fertility problems in mammals, and identify inhibitors, enhancers, antagonists and analogues of GDF-9 which can be used, for example, to diagnose and/or treat fertility problems in mammals.
  • Figure 1 is a bar graph of the results of a progesterone radioimmunoassay showing that conditioned media containing recombinant mouse GDF-9 (mGDF-9) or recombinant human GDF-9 (hGDF-9) stimulated progesterone synthesis by in vitro cultured primary granulosa cells in a time and concentration dependent manner.
  • Figure 2 is a bar graph of the results of a progesterone radioimmunoassay showing that recombinant mGDF-9 stimulated progesterone synthesis by primary granulosa cells in vitro.
  • Figure 3 is a bar graph of the results of a progesterone radioimmunoassay showing that recombinant mGDF-9 with and without fetal calf serum (FCS) stimulated progesterone synthesis by primary granulosa cells in vitro for 24 hours.
  • Figure 4A is a bar graph showing the level of progesterone in the media of granulosa cells cultured for 14 hours in serum-free media in the absence or presence of 50 ng/ml GDF-9 in the presence of varying concentrations of 0, 0.5, or 1 ng/ml FSH; the error bars represent the standard error of the mean (SEM).
  • SEM standard error of the mean
  • Figure 4B is a bar graph showing the level of progesterone in the media after 24 hours of treatment of cells +/- GDF-9 (100 ng/ml) or FSH for triplicate samples in 10% of FCS containing media. *, p ⁇ 0.05 for control versus GDF-9-treated cells cultured without FSH.
  • Figure 5 is a model showing the roles of GDF-9 in the mammalian ovary.
  • FIG. 6 is a summary of the GDF-9 knockout ovary studies. DETAILED DESCRIPTION OF THE INVENTION
  • the present invention relates to a method of identifying an agent which alters (modulates) activity of growth differentiation factor 9 (GDF-9).
  • the method of the present invention is an assay for assessing the ability of an agent to inhibit or enhance the activity of GDF-9, a factor required during early ovarian folliculogenesis.
  • an agent which alters (inhibits, enhances) GDF-9 activity is identified, the following reagents are combined to produce a test sample: cells having a receptor for GDF-9 and a gene whose expression is regulated by binding of GDF-9 to the receptor; GDF-9; and an agent to be assessed.
  • Cells having a receptor for GDF-9 and a gene whose expression is regulated by binding of GDF-9 to the receptor on the cell include, for example, granulosa cells (e.g., mural granulosa cells, cumulus cells) found in the ovarian follicle of mammals.
  • the ovarian follicle of a mammal which includes oocytes surrounded by cells having receptors for GDF-9 (e.g., granulosa cells), in any phase of folliculogenesis can also be used in the methods of the present invention.
  • a primordial, primary, secondary, tertiary (preovulatory) or superovulated ovarian follicle can be used in the methods of the present invention.
  • the ovarian follicle would generally include oocytes, which expresses GDF-9, and granulosa cells. Therefore, the addition of GDF-9 in the test sample is optional in this embodiment.
  • the extent to which the agent to be assessed inhibits the GDF-9 activity or further enhances the activity of GDF-9 present in the ovarian follicle is assessed.
  • Granulosa cells and/or ovarian follicles can be obtained from any mammalian sources as described herein and using skills known in the art. For example, granulosa cells and/or ovarian follicles can be obtained from primate (e.g., monkey, human), bovine, porcine, feline, canine and murine sources.
  • granulosa cells and or ovarian follicles can be obtained from a mammal in any phase of folliculogenesis or from a mammal in a superovulated state.
  • a superovulated state in a mammal can be obtained by administering gonadotropin (e.g., pregnant mare serum gonadotropin) to the mammal, from which granulosa cells and/or ovarian follicles are being obtained.
  • gonadotropin e.g., pregnant mare serum gonadotropin
  • suitable cells for use in the methods of the present invention include cells recombinantly produced to express the GDF-9 receptor and a gene whose expression is regulated by binding of GDF-9 to the receptor on the cell (e.g., recombinantly produced Chinese hamster ovary (CHO) cells which express the GDF-9 receptor and, upon binding of GDF-9 to the receptor, also express hyaluronan synthase).
  • CHO Chinese hamster ovary
  • the methods of the present invention utilize cells having a receptor for GDF-9 and a gene whose expression is regulated by binding of GDF-9 to the receptor on the cell.
  • the gene (one or more) for use in the present invention includes genes which encode, for example, hyaluronan synthase, steroidogenic acute regulatory protein, luetinizng hormone receptor, cyclooxygenase 2, urokinase plasminogen activator, activin/inhibin and follistatin.
  • GDF-9 refers to GDF-9 protein, its individual subunits, multimers of its individual subunits, functional fragments or portions of GDF-9, and functional equivalents and or analogues of GDF-9.
  • functional fragments of GDF-9 are fragments which regulate a gene which encodes, for example, a protein(s) involved in synthesis of hyaluronic acid (e.g., hyaluronan synthase), a protein(s) involved in synthesis of progesterone (e.g., StAR) and/or a protein involved in the synthesis of plasmin (e.g., uPA).
  • GDF-9 functional equivalents or fragments of "GDF-9” include modified GDF-9 protein such that the resulting GDF-9 product has activity similar to the GDF-9 described herein (e.g., regulating a gene which encodes a protein involved in synthesis of hyaluronic acid).
  • GDF-9 suitable for use in the methods and compositions of the present invention can be obtained from a variety of sources or synthesized using skills known in the art.
  • GDF-9 as it occurs in an ovarian follicle can be used in the methods of the present invention.
  • GDF-9 can be purified
  • test sample is maintained under conditions appropriate for binding of GDF-9 to receptors on the cells.
  • the methods described herein is from about 0°C to about 45 °C. In one embodiment, the methods can be performed at about 37 °C, and in another embodiment, the methods can be performed at about 25 °C.
  • a suitable pH range at which the methods can be performed is from about pH 5 to about pH 8, and, in particular, from about pH 7 to about pH 7.4. In one embodiment, the methods can be performed at about pH 7.4.
  • the methods can be performed in a day or over several days. For example, the methods can be performed from about 1 hour to about 96 hours, from about 1 hour to about 48 hours or from about 1 hour to about 24 hours. In one embodiment, the methods can be performed from about 3 hours to about 24 hours. In the methods of the present invention, the extent to which binding of GDF-
  • the extent to which binding of GDF-9 to the receptors on the cells occurs can be determined by directly measurng the particular gene product (e.g., DNA, RNA (mRNA), peptide, protein) produced in response to binding of GDF-9 to the GDF-9 receptor present on the cells used in the assay.
  • the particular gene product e.g., DNA, RNA (mRNA), peptide, protein
  • Examples of such particular gene products include, but are not limited to, hyaluronan synthase, steroidogenic acute regulatory protein, luetinizng hormone receptor, cyclooxygenase 2, urokinase plasminogen activator, kit ligand, activin/inhibin ⁇ B and follistatin.
  • products or functions which occur (e.g., downstream) as a result of the activity of the gene product can also be measured.
  • the gene is involved in the synthesis of hyaluronic acid (e.g., a gene coding for hyaluronan synthase) and a product which results from the activity of the gene, such as hyaluronic acid, can be measured.
  • the gene is involved in synthesis of progesterone (e.g., a gene encoding StAR) and a product which results from the activity of the gene, such as progesterone, phosphorylated StAR and/or another steroid in the progesterone synthetic pathway, can be measured.
  • the gene is involved in the synthesis of plasmin (e.g., uPA) and a product which results from the activity of the gene, such as plasmin and/or a function of the gene, such as the breakdown of plasminogen, can be measured.
  • plasmin e.g., uPA
  • the gene is involved in the production of prostaglandins (e.g., COX-2) and a product which results from the activity of the gene, such as prostaglandins. can be measured.
  • the gene can also be involved in the production of -10-
  • activin B or inhibin B and a product which results from the activity of the gene, such as activin ⁇ B:activin and/or ⁇ B inhibin ⁇ :activin ⁇ B, can be measured.
  • the gene is involved in the production of LH receptor and a product which results from the activity of the gene, such as products produced as a result of the binding of LH receptor (e.g., cyclic AMP), and/or a function which results from the activity of the gene such as the binding of a ligand to the LH receptor, can be measured.
  • LH receptor e.g., cyclic AMP
  • the extent to which binding of GDF-9 in the presence of the agent to be assessed to the receptors on the cells occurs can be determined by measuring the amount of GDF-9 bound to receptors on the cells directly using skills known in the art (e.g., radioreceptor assays).
  • nucleic acids DNA, RNA
  • PCR polymerase chain reaction
  • radiolabeling methods photographing detection of radiolabeled PCR products
  • Southern blots of PCR products Southern blots of PCR products
  • RNase protection assays RNase protection assays
  • Northern blots the presence of protein associated with the gene which is regulated by binding of GDF-9 to the receptor on the cell can be determined using techniques such as high pressure liquid chromatography (HPLC), immunohistochemistry, Western blot analysis and immunoprecipitation.
  • HPLC high pressure liquid chromatography
  • downstream products e.g., hyaluronic acid, progesterone, plasmin
  • the downstream products e.g., hyaluronic acid, progesterone, plasmin
  • HPLC high-density lipoprotein
  • radioimmunoassays for example, HPLC and or radioimmunoassays.
  • the methods of the present invention can further comprise the use of a control sample.
  • a control sample i.e., a sample which includes the same combination of reagents as the test sample except for the agent to be assessed, and which has been processed in the same manner as the test sample.
  • the methods described herein and the agents identified by the methods can be used in a variety of ways.
  • the methods can be used as a screening method to identify agents which are inhibitors, enhancers, antagonists, agonists, and or analogs of GDF-9 and which can be used to diagnose and/or treat reproductive problems or as a diagnostic assay for detecting reproductive problems such as infertility (e.g., an assay of follicular fluid to analyze GDF-9 activity).
  • Agents identified by the methods of the present invention can be used in any disease or condition in which GDF-9 activity is abnormal.
  • agents identified in the methods of the present invention can be used to inhibit fertility (e.g., contraception, contraceptives) or enhance fertility (e.g., increase the success of in vitro fertilization).
  • Granulosa cells in immediate contact with the oocyte in the preovulatory follicle known as cumulus cells, produce a proteoglycan matrix containing hyaluronic acid both in vivo and in vitro in a process called cumulus expansion.
  • This expanded matrix binds the oocyte and cumulus cells together, facilitates follicular extrusion and oviductal fimbria capture, and allows sperm penetration and fertilization (Salustri et al., Zygote, 4:313-315 (1996)). It has been observed that in vitro cumulus expansion depends on follicle stimulating hormone and an oocyte derived factor (Buccione et al., Dev.
  • Cumulus cells stripped away from the oocyte do not expand, assume an adherent, fibroblastic appearance, and produce negligible amounts of hyaluronic acid. If oocytes are added back to the culture, or if the cumulus cells are grown in oocyte conditioned media (OCM) ( ⁇ 1 oocyte/ ⁇ l media), they produce 5-10 fold higher levels of ⁇ 12-
  • mural granulosa cells i.e., granulosa cells of the preovulatory follicle not in contact with the oocyte
  • TGF- ⁇ Stalustri et ⁇ /.N. flzo/. Chem., 265: 19517-19523 (1990)
  • the effect of both OCM and TGF- ⁇ was additive.
  • Anti-TGF- ⁇ antibodies were able to block the TGF- ⁇ effect, but could not block the effect of oocyte conditioned media.
  • TGF- ⁇ itself is not the oocyte-derived signal that stimulates hyaluronic acid production. Additionally, these experiments were repeated using the transcription blocking agent, Actinomycin D, and it was shown that -50% of the effect of TGF- ⁇ was blocked under these conditions (Tirone et al., J. Biol. Chem., 272: 4787-4794 (1997)).
  • HAS2 hyaluronan synthase type 2
  • hCG human chorionic gonadotropin
  • PMSG pregnant mare serum gonadotropin
  • HAS2 Due to its enzymatic activity and its expression pattern, HAS2 is a likely to be responsible for the hyaluronic acid production leading to cumulus expansion, and may be the gene responding to the factor produced by the oocyte or present in oocyte conditioned media.
  • uPA urokinase plasminogen activator
  • recombinant mGDF-9 an oocyte-specific TGF- ⁇ family growth factor
  • This effect on HAS2 expression is specific to GDF-9 as predicted by the follicle culture experiments and not induced by other oocyte-specific TGF- ⁇ family members, (i.e., BMP- 15 and BMP-6).
  • mouse GDF-9 protein is expressed in all oocytes beginning at the type 3a follicle stage including antral follicles.
  • mouse GDF-9 was produced using a Chinese hamster ovary cell expression system.
  • a granulosa cell culture system was established to determine the role of GDF-9 in the regulation of several key ovarian gene products using semi-quantitative RT-PCR.
  • GDF-9 recombinant GDF-9 induced hyaluronan synthase 2 (HAS2), cyclooxygenase 2 (COX-2), and steroidogenic acute regulator protein (StAR) mRNA synthesis but suppressed urokinase plasminogen activator (uPA) and luteinizing hormone receptor (LHR) mRNA synthesis. Consistent with the induction of StAR mRNA by GDF-9, recombinant GDF-9 increased granulosa cell progesterone synthesis in the absence of FSH.
  • HAS2 hyaluronan synthase 2
  • COX-2 cyclooxygenase 2
  • StAR steroidogenic acute regulator protein
  • uPA urokinase plasminogen activator
  • LHR luteinizing hormone receptor
  • GDF-9 functions as an oocyte-secreted paracrine factor to regulate several key granulosa cell enzymes involved in cumulus expansion and maintenance of an optimal oocyte micro environment, processes which are essential for normal ovulation, fertilization, and female reproduction.
  • kits ligand via signaling through c-kit on the oocyte, is directly involved in the increased size of GDF-9-deficient oocytes and the eventual demise of the oocyte.
  • the cells of the GDF-9-def ⁇ cient follicles remain in a steroidogenic cluster which histologically resembles small corpora lutea. However, at the molecular level, these cells are positive for both luteal markers (e.g., LHR and P-450 side chain cleavage) and non-luteal markers (e.g., inhibin and P-450 aromatase).
  • luteal markers e.g., LHR and P-450 side chain cleavage
  • non-luteal markers e.g., inhibin and P-450 aromatase
  • CD-I mice 21 -day old CD-I mice were obtained either from Charles River Laboratories or from the Baylor College of Medicine barrier facility colony. Each female was primed with 7.5 IU pregnant mare serum gonadotropin (PMSG) (0.3 cc of our stock). Initially 44-48 hours later (42-46 hrs later in the last experiment in Example -15-
  • PMSG pregnant mare serum gonadotropin
  • mice were sacrificed by cervical dislocation and ovaries collected removing all fat and bursal tissue possible. Ovaries were placed in prewarmed collection media (DMEM/F12 w/ IX GPS and .3% BSA) in 30 mm dishes. Under the dissecting scope, granulosa cells were released from large antral follicles by repeated puncturing with a 27 Vi gauge needle. Clumps of granulosa cells were dispersed by mouth pipetting up and down through a pulled Pasteur pipette. Oocytes and cumulus-oocyte complexes (COC) were separated from the granulosa cells manually.
  • prewarmed collection media DMEM/F12 w/ IX GPS and .3% BSA
  • granulosa cells were transferred into a clean dish with more prewarmed collection media, and remaining oocytes and COC were removed. Contents of the second dish were transferred into two 1.5 ml eppendorf tubes and spun 3 minutes at 1500 rpm. Fresh collection media was used to wash remaining cells off the plate and placed into the eppendorfs and spun again at 1500 rpm.
  • Granulosa cells were resuspended by gentle pipetting in IX Follicle Culture Medium (alpha MEM, IX Insulin/Transferrin Selenite, IX
  • Glutamine/Penicillin/Streptomycin, 4ng/ml ovine FSH, +/- 10% Fetal Calf Serum 250 ⁇ l-500 ⁇ l per ovary collected; 250 ⁇ l of granulosa cells were plated per well of a 24 well plate. To each well, 250 ⁇ l more IX follicle culture medium with a 2X amount of the treatment to be studied. Final culture volume was 500 ⁇ l.
  • mGDF-9 50 ⁇ l concentrated CHO 19A12 media from mGDF-9 expressing cells + 450 IX F.C.M.
  • Final dilution 1 :20.
  • Estimated final mGDF-9 concentration 50 ng/ml.
  • mBMP-15 lOO ⁇ l concentrated CHO 21A5 media from mBMP-15 expressing cells + 400 ⁇ l IX F.C.M.
  • Estimated final mBMP-15 concentration ⁇ 100 ng ml. -16-
  • RNA Stat-60 (Leedo Medical Supply) was added to each well singly and pipetted up and down to lyse cells. All cells were lysed as verified using the dissecting microscope. To homogenize the cells, cells were pipetted up and down using a P200 pipetman. lOO ⁇ l of chloroform was added to each tube, vortexed, then incubated on ice 10 min. The tubes were spun at full speed for 20 minutes at 4°C. The aqueous phase was carefully collected and RNA was precipitated with 250 ⁇ l isopropanol at -20°C for 2 hours. A very small RNA pellet was observed after spinning 30 minutes at full speed at 4°C.
  • TGGGAGTTGCTGTTGAAGTCGCA (SEQ ID NO: 4) yielding a product of 486 bp.
  • 2 ⁇ l of each RT reaction was used in a standard 50 ⁇ l PCR reaction for each primer set with the following amplification conditions: 3 minutes 94°C, 28 X (1 minute 94°C, 30 seconds 60°C, 1 minute 72°C, and 7 minutes at 72°C). Products were run on a 1.5% agarose gel and visualized.
  • mGDF-9 and hGDF-9 in both the nonconcentrated and concentrated conditioned media were determined by Western blot.
  • MGDF-9 (cone.) media contained -1 ⁇ g/ml and mGDF-9 nonconcentrated media contained - 0.6 ⁇ g/ml.
  • the hGDF-9 conditioned media contained -1 ⁇ g/ml.
  • the following experiment was done using the nonconcentrated mGDF-9 media diluted to the final concentrations of 10 ng/ml, 50 ng ml, 100 ng/ml.
  • hGDF-9 conditioned media was tested at 50 ng/ml, 100 ng/ml and 300 ng ml. The purpose was to define an active concentration range and an early timecourse for effect.
  • the IX follicle culture media did not contain fetal calf serum, so all effects seen are serum independent.
  • yeast tRNA was added to each sample during the homogenization step.
  • ⁇ P 32 -dCTP was added to each PCR reaction. Only l ⁇ l of RT reaction was used as template and amplification occurred for 20 cycles. 5 ⁇ l of each reaction was run on a 4% polyacrylamide gel, the gel was dried and exposed to Kodak X-OMAT autoradiography film.
  • results By 3 hours in culture 100 ng/ml mGDF-9 stimulates HAS2 production and inhibits uPA production compared to the CHO control, whereas 100 ng/ml of hGDF-9 fails to stimulate HAS2 and significantly induces uPA. After 5 hours in culture 10 ng/ml of mGDF-9 induces HAS2, while 50 ng/ml and 100 ng/ml both induce an approximately 5-10 fold higher level. After 5 hours in culture 100 ng/ml of hGDF-9 induces a detectable HAS2 signal, while 300 ng/ml induce a level approaching that of 50 ng/ml of mGDF-9.
  • mGDF-9 inhibits uPA expression, while all concentrations of hGDF-9 appear to have a stimulatory effect.
  • GDF-9 regulation of uPA may be dependent on a GDF-9 concentration gradient in the antral cavity generated by diffusion of GDF-9 from the oocyte, with lowest levels reaching the mural granulosa cells which are the furthest from the oocyte, defining where follicle wall breakdown for ovulation will occur.
  • mGDF-9 is the oocyte-derived growth factor that normally causes hyaluronic acid production and inhibition of uPA in the cumulus cells. Additionally, these results indicate that granulosa cells can respond to GDF-9 and express the GDF-9 receptor. See the Table.
  • the progesterone RIA kit was obtained from Diagnostic Products Corporation and detects progesterone levels from 0.1 -40 ng/ml using a 100 ⁇ l sample volume. Higher concentrations can be measured by diluting the experimental sample before assaying with the provided dilution solution.
  • Recombinant mGDF-9 stimulated progesterone synthesis by in vitro cultured primary granulosa cells in a time and concentration dependent manner (See Figures 1, 2 and 3). At 5 hours, 60 ng/ml of mGDF-9 showed a 2.5 fold increase and 300 ng/ml showed a 4.5 fold increase (see Figure 2). This stimulation of progesterone is also seen at 24 hours where a 3.5 to 4 fold stimulation was observed (See Figure 3).
  • the rate limiting step in progesterone synthesis is the catalysis of cholesterol to pregnenolone by the enzyme complex cytochrome P450 side chain cleavage (sec).
  • Non-luteinized cultured rat granulosa cells can be stimulated to increase P450scc mRNA levels and to produce progesterone with forskolin, a cAMP agonist, in vitro (Oonk et al, J. Biol. Chem., 264:21934-21942) indicating that progesterone synthesis at this stage is cAMP-dependent and is associated with increases in
  • mice GDF-9 cDNA (Incerti, B., et al, Biochim. Biophys. Ada., 7222:125-128 (1994)) was subcloned into the expression vector pHTop containing the processing gene PACE (a gift from Dr. Monique Davies, Genetics Institute, Cambridge, Massachusetts).
  • the GDF-9 expression vector was lipofectin transfected into CHO cells under standard conditions (Gibco BRL Life Technologies). Expression of mouse GDF-9 in CHO cells was subsequently driven by a tet-regulatable promoter while an SV40 promoter regulated expression of PACE. Stable, positive clones were selected in the presence of 0.02 ⁇ M methotrexate in ⁇ -modified eagles' medium ( ⁇ MEM) containing 10% heat -23-
  • GDF-9-expressing cells were incubated for 24 hours in Opti-MEM reduced serum collection media containing 100 mg/ml heparin (SIGMA, St. Louis, MO). The media were harvested, and GDF-9 protein levels were determined by SDS-polyacrylamide gel electrophoresis (PAGE) with subsequent immunoblotting (see next section).
  • Mouse antihuman GDF-9 monoclonal antibody (described above) was used at a 1 : 1000 dilution in blocking solution, and an anti-mouse secondary antibody conjugated to horseradish peroxidase (Southern Biotechnology Associates, Birmingham, AL) was used at a 1 :2500 dilution in blocking solution.
  • Several batches of recombinant mouse GDF-9 were produced during the course of these studies, all of which appeared to have similar activities based on Western blot quantitation (i.e., immunoreactivity correlated with bioactivity). -24-
  • ICR mice 21-24 day-old female CD-I (ICR) mice (Baylor College of Medicine) mice (Baylor College of Medicine) were injected with 7.5 IU Gestyl (Diosynth, B.V., Holland) and ovaries were harvested 44-48 hours later, dissected free of fat and surrounding tissue, and placed in minimal essential media with 25mM HEPES supplemented with 0.3 mg/ml L-glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin (Gibco BRL, Grand Island, NY), and 0.3% BSA (SIGMA).
  • Mural granulosa cells were released by puncturing large antral follicles.
  • GCM 2X granulosa cell culture media
  • GDF-9-containing media or control conditioned media were diluted to 2X the final concentration in ⁇ -MEM. Equal volumes of 2X GDF-9-containing media or control media were combined with granulosa cells in 2X culture media and cultured at 37 °C in a humidified atmosphere with 5% C0 2 . After varying periods of culture, non-adherent cells were pelleted from the media, and the media were stored at -20 °C. Granulosa cells were lysed and total RNA was isolated using RNA Stat-60 (Leedo Medical Laboratories, Houston, TX) following manufacturer's protocol.
  • a PCR product of 403 bp is amplified from RNA, easily distinguished from amplification of contaminating DNA.
  • Mouse uPA mRNA expression was detected using 5' GTTCAGACTGTGAGATCACTGG 3' (SEQ ID NO: 7) (sense) and 5' CAGAGAGGACGGTCAGCATGG 3' (SEQ ID NO: 8) (antisense) primers that span two introns of 1.4 kb total length.
  • a PCR product of 434 bp is amplified from RNA.
  • Mouse hypoxanthine phosphoribosyltransferase was amplified using 5' CCTGGTTAAGCAGTACAGCC 3' (SEQ ID NO: 5) (sense) and 5 ACTAGGCAGATGGCCACAG 3 * (SEQ ID NO: 6) (antisense) primers which span three introns of unknown sizes and gives an expected mRNA-derived product size of 309 bp from RNA.
  • Mouse StAR mRNA expression was detected using 5' TCGCTTGGAGGTGGTGGTAGAC 3' (SEQ ID NO: 9) (sense) and 5' GCAGGTCAATGTGGTGGACAGT 3' (SEQ ID NO: 10) (antisense) primers which span multiple small introns and give an mRNA-derived 522 bp product.
  • Mouse cholesterol side chain cleavage P-450 mRNA expression was detected using 5' GCCAACATTACCGAGATGC 3' (SEQ ID NO: 11) (sense) and 5' CGAACACCCCAGCCAAAGCC 3' (SEQ ID NO: 12) (antisense) primers and give an mRNA-derived 426 bp product.
  • Mouse COX-2 mRNA expression was detected using 5' CTCCTTTTCAACCAGCAGTTCC 3' (SEQ ID NO: 13) (sense) and 5' TCTGCAGCCATTTCCTTCTCTC 3' (SEQ ID NO: 14) (antisense) primers and give a 377 bp product.
  • Mouse LH receptor mRNA expression was detected using 5' CTTATACATAACCACCATACCAG 3' (SEQ ID NO: 15) (sense) and 5' ATCCCAGCCACTGAGTTCATTC 3' (SEQ ID NO: 16) (antisense) primers which span multiple introns and give a 516 bp product.
  • PCR products amplified from granulosa cell cDNA were initially isolated, subcloned, and sequenced to confirm that they matched published sequences.
  • [ ⁇ 32 P]-dCTP was added to each PCR reaction and products were separated by electrophoresis on a 4% polyacrylamide gel. The gels were dried and exposed to autoradiography, and radioactive bands quantitated on a Molecular Dynamics phosphorimager (Storm 860).
  • Probes for HAS2 and uPA were generated from the aforementioned subcloned PCR products by random priming with [ ⁇ 32 P]-dATP using the StripEZ probe synthesis kit (Ambion, Austin, TX). The membrane was hybridized, washed, and subjected to autoradiography as described (Mahmoudi, M. and Lin, V.K., Biotech., 7:331-332 (1989)). The probe was removed from the membrane using the Strip-EZ removal reagents (Ambion) following the manufacturer's protocol. The same blots were then reprobed with glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) as a loading control. Signals for each probe were quantitated on a Molecular Dynamics phosphorimager.
  • GPDH glyceraldehyde-3 -phosphate dehydrogenase
  • Progesterone in the culture media was measured in duplicate by a specific, solid-phase radioimmunoassay using a kit from Diagnostic Products Corporation (Los Angeles, CA) according to the manufacturer's instructions.
  • the sensitivity of this assay is 0.02 ng/ml, and calibration standards between 0. 1 and 40 ng/ml were used.
  • Cumulus cell-oocyte complexes were collected as described above. The oocyte was removed from each complex using a micro injection apparatus as previously described (Buccione, R., et al, Dev. Biol, 138:16-25 (1990)). Successful oocytectomy was assessed by the removal of the germinal vesicle along with the majority of ooplasm. Oocytectomized complexes were incubated for 18 hours in groups in 20 ⁇ l droplets of granulosa cell culture media supplemented with 10% fetal bovine serum and 5 ng/ml or 100 ng/ml of oFSH with or without 1 ⁇ g/ml oLH -27-
  • GDF-9 Immunohistochemical detection of GDF-9 in mouse ovaries. Using a monoclonal antibody to human GDF-9, GDF-9 protein was specifically detected in mouse oocytes. At low power magnification of an immunohistochemically-stained ovary, GDF-9 immunoreactivity was detected only in oocytes, whereas oocytes in GDF-9-deficient ovaries did not stain. Primordial (type 2) oocytes were negative consistent with the absence of GDF-9 mRNA expression (McGrath, S.A. et al, Mol. Endocr., 9:131-136 (1995)) and Elvin and Matzuk, unpublished data).
  • GDF-9 immunoreactivity was first seen at low (and variable) levels in oocytes of type 3a follicles (follicles with less than 20 cuboidal granulosa cells arranged in one concentric layer around the oocyte) and was higher in the oocytes of type 3b follicles and beyond.
  • Full-grown oocytes of multilayer preantral follicles consistently stained more intensely for GDF-9, and GDF-9 immunoreactivity was clearly detected in oocytes of cumulus cell-oocyte complexes of large antral and pre-ovulatory follicles.
  • GDF-9 immunoreactivity was excluded from the germinal vesicle (i.e., nucleus).
  • GDF-9 Chinese hamster ovary (CHO) cells were stably transfected with an expression vector containing both a full-length mouse GDF-9 cDNA and a cDNA for PACE, a prepropeptide sequence cleaving enzyme. -28-
  • the mature GDF-9 sequence contains a single N-linked glycosylation site (Asn 325 -Leu 326 - Ser 327 ).
  • the predominant 21 kD form would correspond to the cleaved, mature monomeric form of mouse GDF-9 with one N-linked oligosaccharide.
  • This band ran at an identical position as the recombinant human GDF-9 synthesized in CHO cells.
  • the band at 60 kD corresponded to the glycosylated, unprocessed (prohormone) form (441 amino acids) which is also secreted into the media.
  • GDF-9-containing medium was treated with N-glycanase to remove the N-linked oligosaccharides.
  • This treatment reduced the size of the 21 kD band to 16 kd, the same molecular weight as the bacterially-produced GDF-9 mature peptide, and also reduced the 60 kD band to 50 kD. Since the 21 kD glycosylated GDF-9 form is always the most abundant form, this strategy to produce recombinant, glycosylated GDF-9 in mammalian cells in the presence of PACE and under serum free culture conditions is efficient.
  • HAS2 hyaluronan synthase 2
  • uPA uPA mRNA synthesis by recombinant GDF-9.
  • HAS2 Hyaluronan synthase 2
  • HAS2 and uPA oocyte-secreted factor responsible for inducing cumulus expansion through increased hyaluronic acid matrix synthesis and decreased hyaluronic acid matrix degradation
  • RT-PCR incorporation of radiolabeled nucleotides into specific products was used to monitor the expression levels of HAS2 and uPA in control and GDF-9-treated granulosa cell cultures.
  • a linear range of product amplification for each oligonucleotide pair of the three genes i.e., HAS2, uPA, and HPRT
  • GDF-9-induced peak level indicating a very low level of basal activity in these cells.
  • the time course for uPA expression in control granulosa cells cultured in the absence of GDF-9 indicated that uPA levels increase over the first 9 hours in culture.
  • granulosa cells treated with 100 ng/ml of recombinant GDF-9 maintained a detectable but much lower level of uPA expression.
  • the band intensity from the GDF-9-treated sample was -15% of the control, and at 9 hours, it was -9% of the control treated sample band intensity (normalized to HPRT for each sample).
  • HAS2 and uPA expression were examined by Northern blot analysis in the presence or absence of 50 ng/ml of GDF-9 .
  • Total RNA from each sample (at 0 or 5 hours incubation in the presence or absence of 50 ng/ml GDF-9) was subjected to Northern blot analysis and hybridized with either an HAS2 or uPA probe and subsequently with a GAPDH probe.
  • the signals were quantitated on a phosphorimager and HAS2 and uPA levels and normalized to GAPDH.
  • HAS2 was barely detectable in mural granulosa cells at 0 hours or after 5 hours of culture in the control sample.
  • both the 4.8 kb and 3.2 kb HAS2 mRNA forms (Spicer, A.P., et al, J. Biol Chem., 277:23400-23406 (1996)) were increased 9.7-fold compared to control.
  • Northern blot analysis of uPA showed that 50 ng/ml GDF-9 suppressed uPA synthesis to 40% of control cultures.
  • the Northern blot data confirmed our RT-PCR analyses.
  • Recombinant GDF-9 causes cumulus expansion of oocytectomized cumulus cell- oocyte complexes.
  • Intact cumulus cell-oocyte complexes were isolated from PMSG-treated immature female mice. Using a transgenic micro manipulator set-up, the oocytes from these complexes were punctured, and the oocyte contents were suctioned. -31-
  • oocytectomized cumulus complexes were spherical objects approximately 100 ⁇ m in diameter consisting of several layers of granulosa cells that surround an empty zona pellucida. After 18 hours in culture, cumulus cells from 25 out of 25 oocytectomized complexes cultured in control media (i.e., deficient in GDF-9 but containing 10% fetal calf serum and 5 ng/ml or 150 ng/ml of FSH) adhered to the tissue culture plate and assumed a fibroblastic appearance.
  • control media i.e., deficient in GDF-9 but containing 10% fetal calf serum and 5 ng/ml or 150 ng/ml of FSH
  • GDF-9 protein is first detected at low levels within growing oocytes of primary follicles (type 3a follicles), is present at higher levels in full-grown oocytes of type 3b follicles, and is detected in oocytes of every subsequent developmental stage.
  • GDF-9 protein is also synthesized by oocytes of large antral and preovulatory follicles in which the oocyte is closely associated with cumulus cells.
  • the GDF-9 protein is detected in oocytes of type 3a follicles, it only becomes essential for folliculogenesis at the type 3b-type 4 follicle transition. This indicates that the GDF-9 signal transduction cascade is not active prior to the type 3b follicle stage.
  • GDF-9 could substitute for oocytes and oocyte-conditioned media in assays analyzing HAS2 induction and uP A suppression typical of processes occurring in pre-ovulatory follicles.
  • other oocyte-expressed TGF- ⁇ family members, BMP- 15 and BMP-6 were unable to substitute for GDF-9 in these granulosa cell assays.
  • Mural granulosa cells, isolated from antral follicles treated with recombinant GDF-9, were induced to express HAS2 in a dose-dependent and time dependent manner.
  • GDF-9 induced approximately 10-fold higher levels of HAS2 mRNA in mural granulosa cells which corresponds well to the maximum effect of oocytes on HA synthesis. Additionally, the dose-response curve for GDF-9 was very similar to that of the oocyte-conditioned media.
  • Very low doses e.g., 0.5 oocytes/ ⁇ l or 10 ng/ml GDF-9 induced very low but detectable increases in hyaluronic acid synthesis or HAS2 expression whereas 1 oocyte/ ⁇ l or 30-50 ng/ml GDF-9 caused a much more dramatic induction which plateaus at 2-4 oocytes/ ⁇ l or 120-300 ng/ml GDF-9 (Salustri, A., et al, Dev. Biol, 138: 26-32 (1990)).
  • the time course of GDF-9 action also agreed with previous data for the oocyte-produced factor.
  • HAS2 mRNA was induced by 2 hours in culture with GDF-9, oocyte-induced hyaluronic acid became detectable at low levels after 2.5 hours (Salustri, A., et al, J. Biol. -34-
  • the data described herein indicates that the difference in the in vivo phenotype of mural granulosa cells versus expanding cumulus granulosa cells is not intrinsic to the cells themselves but is due to their proximity to the oocyte and the concentration gradient of the oocyte-produced GDF-9.
  • the conversion of cholesterol to pregnenolone is the rate-determining step in granulosa cell steroidogenesis.
  • the rate of pregnenolone synthesis depends on the level and activity of the reaction catalyzing enzyme, cytochrome P-450 side chain cleavage (P-450scc), and its access to its substrate cholesterol via stimulation of the steroidogenic acute regulatory protein (StAR) (Rennert, H., et al., "Intracellular cholesterol dynamics in steroidogenic cells", In: L.C. Adashi, EN. (ed) 77ie Ovary, Raven Press, Ltd., New York, pp. 1470164 (1993)).
  • StAR steroidogenic acute regulatory protein
  • the data described herein confirms that FSH stimulates P-450scc mRNA synthesis in mouse granulosa cells but demonstrates that GDF-9 did not significantly affect P-450scc mRNA synthesis.
  • FSH has only a small inductive effect on StAR mRNA, but GDF-9 with or without FSH significantly induces StAR expression. Consequently, both GDF-9 and FSH can independently increase production of progesterone by the granulosa cells and appear to function in the same pathway but via different mechanisms.
  • in situ hybridization analysis of LH receptor in preovulatory follicles demonstrates that LH receptor is suppressed in the cumulus cells but not the mural granulosa cells, whereas after LH treatment in vivo, COX2 expression is highest in the cumulus cells.
  • recombinant GDF-9 suppresses LH receptor mRNA but induces COX-2 expression, mimicking the normal expression of these genes in the cumulus cells.
  • Eppig and colleagues Eppig, JJ. et al, Biol. Reprod., 56:976-984 (1997) and Eppig, J.J. et al, Mol. Reprod.
  • GDF-9 can stimulate changes in cell morphology, gene expression, and steroid production indicating that granulosa cells, at least from primary follicles and from antral follicles, possess receptors which bind GDF-9 (see Figure 5 for a summary of the findings described herein). Since GDF-9 dramatically increases the level of COX-2 expression, it is -36-
  • GDF-9 precursor is only processed to an active mature dimer at the type 3b stage and at the antral follicle stage.
  • the regulation of the GDF-9 processing enzyme would be one way to regulate the activity of GDF-9 post-translationally.
  • GDF-9-regulated and GDF-9-independent transcription factors function together to regulate the synthesis of COX-2.
  • At least one regulator of COX-2 is the transcription factor enhancer-binding protein P (C/EBP ⁇ ).
  • C/EBP ⁇ which is induced between 4 and 7 hours after hCG treatment, binds to the COX-2 promoter to downregulate COX-2 mRNA expression.
  • COX-2 expression in the ovary normally peaks at 4 hours after hCG treatment while COX-2 protein continues to be present in the cumulus cells after ovulation (Lim, H., et al, Cell, 97:197-208 (1997)).
  • COX-2 knockout mice are infertile due to defects in ovulation and impaired oocyte maturation (Lim, H., et al. Cell,
  • the membrane was hybridized, washed, and subjected to autoradiography as described (Mahmoudi, M. and Lin, V.K., Biotech., 7: 331-332 (1989)).
  • the probe was removed from the membrane using the Strip-EZ removal reagents (Ambion) following the manufacturer's protocol.
  • the same blots were then reprobed with gl yceraldehyde-3-phosphate dehydrogenase (GAPDH) or 18S ribosomal RNA as a loading control. Signals for each probe were quantitated on a Molecular Dynamics photodensitometer. -38-
  • Hybridization was carried out at 55°C with 5 x 10 6 cpm of each riboprobe per slide for 16-18 hrs in 50% deionized formamide/0.3M NaCl/20mM TrisHCl (pH 8.0)/5 mM EDTA/10 mM NaPO4 (pH 8.0)/10% Dextran sulfate/lX Denhardt's/0.5 mg/ml yeast RNA. High stringency washes of 2X SSC/50% formamide and 0.1XSSSC at 65 °C were carried out.
  • Dehydrated sections were dipped in NTB-2 emulsion (Eastman Kodak, Rochester, NY) and exposed 2-14 days, depending on the probe, at 4°C. After developing, the slides were counterstained with hematoxylin and mounted for photography.
  • Ki-67 staining sections were steamed for 35 minutes in 0.1 M citrate buffer pH 6.0.
  • p27 staining sections were steamed for 35 minutes in pH 8.0 Tris-EDTA antigen retrieval solution.
  • Ki-67, p27 and P-450scc all sections were blocked for 30 minutes in IX PBS with 0.05% Tween-20, 2% normal mouse serum (Sigma) and 2% normal goat serum, and incubated in the primary antibody for 1 hour at room temperature.
  • PCNA detection was accomplished using the Super Sensitive Mouse Antibody Animal Detection kit (Biogenex, San Ramon, CA) containing anti-mouse IgG biotinylated secondary antibody preabsorbed with rat tissue.
  • P-450scc, p27, and Ki-67 antibodies were detected using the Super Sensitive Rabbit Antibody Detection kit (Biogenex) containing anti-rabbit IgG biotinylated secondary antibody preabsorbed with mouse tissue.
  • PCNA and P-450scc were detected using strep tavidin-conjugated alkaline phosphatase label and New Fuschin substrate (Biogenex) while p27 was detected with strep tavidin-conjugated horseradish peroxidase label (Biogenex) and DAB substrate (Vector Laboratories).
  • Ovaries were stained for apoptotic cells by a modified TUNEL method using the Apoptag Plus Complete Apoptosis Detection kit (Oncor Laboratories, Gaithersburg, MD) following the manufacturer's instructions. Nuclei were counterstained with Propidium iodide/ Antifade mounting media (Oncor Laboratories).
  • Oligo-dT primed cDNA from 1 ⁇ g of either control or GDF-9-def ⁇ cient ovarian RNA was synthesized using Superscript reverse transcriptase (GibcoBRL) following the manufacturer's protocol. 1 ⁇ l of each RT reaction (1/20 of total) was used in each 25 ⁇ l PCR reaction primed with kit ligand-specific oligonucleotides: 5*CCAGAAACTAGATCCTTTACTCCT 3' (SEQ ID NO: 17) (sense - nts 493-517 of S40364) and 5' CTGTTGCAGCCAGCTCCCTTAG 3' (SEQ ID NO: 18) (antisense 943-919 of S40364) primers which span introns and an 84 bp alternatively spliced exon. Amplification of the KL-1 form yields a product of 450 -41-
  • PCNA Proliferating cell nuclear antigen
  • Ki-67 a component of the granular nucleolus, is expressed in all cell cycle phases except GO (Gerdes, J., et al, J. Immunol, 755:1710-1715 (1984)).
  • the most highly proliferative granulosa cells are found in the wild-type antral follicle, in which the majority of the granulosa cells are PCNA and Ki-67 positive.
  • type 3b (large one layer) and type 4 (2 layer) follicles of wild-type ovaries immunohistochemical analysis of PCNA or Ki67 showed that >50% of the granulosa cells in the cross-sections are positive (i.e., positive defined as intense red staining of the nucleus; negative defined as light or diffuse staining of the nucleus and cytoplasm).
  • type 3b follicles with intact oocytes in the GDF-9-deficient ovary demonstrated ⁇ 10% positive staining granulosa cells/cross-section indicating that nearly all of the granulosa cells are blocked at GO.
  • p21 and p27 are well documented inhibitors of the cell cycle, and are correlated with cell cycle arrest upon luteinization in the ovary (Robker, R.L. and Richards, J.S., Biol. Reprod., 59:476-482 (1998)). Based on the relative lack of proliferation of the granulosa cells in the GDF-9 knockout ovaries, expression of both p21 and p27 mRNA was examined by in situ hybridization and p27 protein by immunohistochemistry.
  • p21 mRNA was detected at low levels ubiquitously in both wild-type and GDF-9-deficient ovaries with higher levels in wild type atretic follicles and scattered cells in the corpora lutea, and in the luteinized follicular nests of the GDF-9 deficient ovary.
  • p27 mRNA was also expressed ubiquitously at low levels throughout the ovary but was more abundant in the corpora lutea of wild-type ovaries.
  • granulosa cells of the one-layer follicles expressed detectable levels of p27 message, while small groups of cells in the center of the GDF-9-deficient ovary expressed higher levels.
  • nuclear p27 immunoreactivity was clearly detectable in the majority of luteinized granulosa cells within the wildtype corpus luteum, and within the luteinized follicular nests of the GDF-9-deficient ovary.
  • Reduced p27 nuclear staining was also present in granulosa cells of both wildtype and GDF-9-deficient one-layer follicles, which was clearly higher than the staining in the negative control or in the interstitial cells.
  • the c-kit kit ligand-signaling pathway has been shown to be important for germ cell proliferation and folliculogenesis (Besmer, P., et al, Dev. Suppl, 125-137 (1993); Huang, E.J. et al, Dev. Biol, 757:100-109 (1993) and Kuroda, H., et al, Dev. Biol, 726:71-79 ( 1988)).
  • By northern blot analysis it was shown that c-kit mRNA is expressed in GDF-9-deficient ovaries and that levels are comparable to or -44-
  • c-kit mRNA was localized to the oocyte and theca-interstitial cells of the wild-type ovary, but was excluded from granulosa cells as previously demonstrated (Manova, K, et al, Devel, 770:1057-1069 (1990)). In the GDF-9-deficient ovaries, c-kit mRNA localized only to oocytes, with only background levels of silver grains present over other cell types.
  • kit ligand expression in GDF-9-def ⁇ cient ovaries was increased 32-fold compared to expression in wild-type ovaries.
  • IGF-1 which is also expressed in the granulosa cells of early preantral follicles, however, did not show a similar increase in expression in the
  • kits ligand represented a specific regulatory interaction, rather than a tissue composition effect.
  • in situ hybridization is not a reliable method for quantitating mRNA expression
  • relative expression levels between wildtype and GDF-9-deficient ovaries can be compared by positioning sections from both types of ovaries close together on the same slide to minimize inter-slide variability in hybridization efficiency and emulsion thickness.
  • kit ligand expression was barely detected in the wild-type ovary, with a faint signal above background apparent in granulosa cells of preantral follicles.
  • granulosa cell expression of kit ligand was abundant.
  • Type 3a and early type 3b follicles had detectable levels of kit ligand, while the largest one-layered follicles showed more intense staining.
  • kit ligand was somewhat easier to detect in these immature ovaries due to the increased number of preantral follicles, the relative expression level per follicle was never comparable to the level seen in the GDF-9-deficient ovaries at similar ages.
  • Kit ligand expression was increased further in the granulosa cells in asymmetric follicles, which are presumably destined to undergo oocyte degeneration. However, soon after the oocyte degenerated, kit ligand expression disappeared, and was also absent in the follicular nests. -45-
  • KL-1 and KL-2 there are two alternatively-spliced forms of KL, KL-1 and KL-2, which differ by 84 bp.
  • This alternative splicing results in an additional 28 amino acids in KL-1, which includes a proteolytic cleavage site. Since membrane-bound KL is more active than free KL, KL-2, the more stable, cell-associated form is consequently more potent (Besmer, P., et al, Dev. Suppl, 125-137 (1993)).
  • RT-PCR By non-quantitative RT-PCR using primers that can distinguish KL-1 from KL-2, both forms of KL were detected in both the wild-type and GDF-9-deficient ovaries.
  • TGF- ⁇ superfamilly members (Activins, Inhibins, Follistatin)
  • GDF-9-deficient versus wild-type ovaries In wild-type ovaries, inhibin ⁇ was expressed in granulosa cells of all growing follicles (type 3a through the preovulatory stage), but was excluded from corpora lutea. In GDF-9-deficient ovaries, inhibin ⁇ was expressed highly in the one-layer follicles, in the follicles with degenerating oocytes, and in the central steroidogenic follicular nests.
  • ⁇ A message was not detectable in the one-layered follicles, weakly localized to granulosa cells of follicles with degenerating oocytes, and was expressed at high levels in oocyte-deficient follicular nests.
  • the levels of ⁇ A expression were equivalent between GDF-9-deficient and wild-type ovaries.
  • FSH receptor FSH receptor
  • aromatase cytochrome P-450 aromatase
  • ER ⁇ estrogen receptor ⁇
  • FSHR By in situ hybridization in the wild-type ovary, FSHR, ER ⁇ , and cytochrome aromatase were detected specifically in the granulosa cells as previously reported (Richards, J.S., Endocrine Reviews, 75:725-751 (1994); Byers, M., et al., Mol. Endocr., 11 : 172-182 (1997) and Camp T., et al, Mol. Endocr., 5:1405-1417 (1991)).
  • ER ⁇ was expressed at low levels in one-layer follicles, and at higher levels in multilayer follicles.
  • FSHR was expressed in multilayer preantral and antral follicles.
  • Aromatase which is normally induced by FSH stimulation of the granulosa cells, was expressed at high levels specifically in the pre-ovulatory follicle.
  • ER ⁇ was -47-
  • GDF-9-deficient ovaries contain multiple, centrally located nests of cells that have the appearance of luteinized granulosa cells. By electron microscopic analysis, the cells of these nests contain multiple lipid droplets and mitochondria with tubular cristae typical of highly steroidogenic cells (Dong, J., et al, Nature, 555:531-535 (1996)).
  • COX-2 cyclooxygenase 2
  • LHR LH receptor
  • P-450scc cholesterol side chain cleavage cytochrome P-450 protein
  • RNA from superovulated ovaries showed three distinct bands, while no bands could be detected in the unstimulated wild type or GDF-9-deficient ovary lanes. Consistent with the northern blot data, in situ hybridization showed that in wild-type ovaries stimulated with PMSG and hCG,
  • COX-2 is expressed by the granulosa cells of pre-ovulatory follicles (Sterrieck, E., et al, Genes Dev., 77:2153-2162 (1997)). The highest expression at 5 hours occurred in the cumulus cells of the wild type ovary, while COX-2 expression was completely undetectable in the GDF-9-deficient ovary. In contrast to the above-mentioned COX-2 expression data, it has been previously shown that LHR is expressed in the GDF-9-deficient ovary at levels -48-
  • LHR was expressed by theca cells, granulosa cells of preovulatory follicles and luteinized granulosa cells of corpora lutea (Sirois, J., et al, J. Biol. Chem., 267:11586-11592 (1992)).
  • granulosa cells of nonluteinized and luteinized follicular nests expressed LHR at very high levels.
  • P-450scc protein Stimulation of theca and luteinized granulosa cells by LH stimulates production of the steroidogenic enzyme P-450scc and subsequent synthesis of progesterone (Richards, J.S., Endocrine Reviews, 75:725-751 (1994)).
  • P-450scc protein was present in theca cells, corpora lutea, and secondary interstitial tissue.
  • P-450scc protein was detected at low levels in nonluteinized follicular nests and at much higher levels in the steroidogenic "luteinized" follicular nests.
  • mice Female 6 week old GDF-9-deficient mice had average serum progesterone levels of 3.4 ng/ml, compared to 2.6 ng/ml in wild-type mice, indicating that these nests are not only capable of expressing markers but also functioning like "miniature" corpora lutea. However, as mentioned earlier, these follicular nests also express inhibin ⁇ , a marker that is normally never observed at significant levels in corpora lutea.
  • GDF-9 mRNA McGrath, S.A., et al, Mol. Endocr., 9:131-136 (1995)
  • protein Elvin, J.A., et al, Mol. Endocr., (Submitted) (1999)
  • GDF-9 knockout mice show a block at the type 3b primary follicle stage.
  • the granulosa cells of the type 3b follicle essentially lay dormant; neither cell division nor apoptosis is observed in the granulosa cells of the follicles until the oocyte is lost.
  • GDF-9 protein is synthesized at the type 3 a stage, the GDF-9 signal transduction cascade must only become essential at the type 3b stage for further follicular growth.
  • these studies suggest that recruitment of primordial follicles and growth of the granulosa cells from the -49-
  • primordial follicle stage ( ⁇ 20 granulosa cells) to the type 3b stage (90 granulosa cells)
  • Pedersen, T. "Follicle Growth in the Mouse Ovary", In: S. A. Bigger JD (ed) Oogenesis, University Park Press, Baltimore, pp. 361-376. (1972)
  • GDF-9 GDF-9 knockout ovary
  • GDF-9 may be required to induce competence to respond to pro-apoptotic stimuli.
  • FSH farnesose
  • the elevated serum FSH cannot overcome the type 3b block, it could promote granulosa cell survival.
  • the granulosa cells in the GDF-9-deficient ovaries may bypass this "apoptosis-competent" state by differentiating after the oocyte degenerates to form the steroidogenic follicular nests.
  • GDF-9 is an important factor for the "differentiation" of the granulosa cells, allowing the post-type 3b granulosa cells to acquire specific characteristics such as the capability to undergo apoptosis.
  • kits ligand expression by a paracrine mechanism
  • kit ligand expression In gonadotropin-stimulated mice, there is a gradient of kit ligand expression whereby granulosa cells farthest from the oocyte (i.e., mural granulosa cells) express the highest levels while those closest to the oocyte (i.e., cumulus cells) express very low or undetectable levels (Motro, B. and Bernstein, A., Dev. Dyn., 197:69-79 (1993)).
  • kit ligand that action of other oocyte-produced and extrafollicular factors unopposed by GDF-9 contribute to the increased kit ligand expression observed herein.
  • the kit ligand that is produced in the GDF-9-deficient ovaries also appears to be active. GDF-9- deficient ovaries contain significantly more mast cells per section, likely due to kit ligand stimulated increased recruitment and proliferation as has previously been reported in other systems. In addition, kit ligand has also been shown to stimulate oocyte growth in vitro (Packer, A. I., et al, Dev. Biol, 767:194-205 (1994)).
  • the oocytes in the GDF-9-knockout ovary grow more rapidly and to a 15% greater maximum size compared to the controls (Carabatsos, M., et al., Dev. Biol., 203:373- 384 (1998)), before ultimately degenerating, providing further evidence of functional granulosa cell derived kit ligand signaling through c-kit on the oocyte. As demonstrated herein by Northern blot analysis, other members of the TGF- ⁇ superfamily continue to be expressed in the GDF-9-deficient ovary.
  • inhibin ⁇ and activin ⁇ A subunits are expressed in GDF-9-deficient ovaries at similar levels to controls, whereas activin ⁇ B and follistatin are dramatically decreased.
  • In vitro activin A has been shown to stimulate follicular growth (Mather et al., 1997) and to enable FSH stimulation of granulosa cell DNA synthesis (Miro, F. and Hillier, S., Endocr., 137: 464-468 (1996)).
  • activin A + FSH stimulate granulosa cells from immature follicles to produce progesterone, but decreased progesterone synthesis by granulosa cells from differentiated culture with or without FSH (Miro, F., et al., Endocr., 129:3388-3394 (1991)).
  • FSH FSH-maleimidomase
  • he activin effect in the GDF-9-deficient ovaries is enhanced by the reduced level of follistatin, an activin binding protein and antagonist.
  • Example 5 Identification of a stimulatory effect of recombinant mGDF-9 on progesterone synthesis by in vitro cultured mural granulosa cells.
  • the effect of mGDF-9 on progesterone production occurs only in the absence of or at low doses (0.5 or 1 ng/ml) of follicle stimulating hormone (FSH).
  • FSH follicle stimulating hormone
  • Example 6 Identification of a stimulatory effect of recombinant mGDF-9 on follistatin and activin ⁇ B expression by in vitro cultured mural granulosa cells.
  • RNA was reverse transcribed into cDNA which was used to generate probes for hybridization to the Affymetrix developmental biology 250 gene chip. Based on the results of this initial screening method, the granulosa cell culture experiment was repeated and RNA was prepared and used for Northern blot analysis.
  • Example 7 Identification of an inhibitory effect of recombinant mGDF-9 on luteinizing hormone receptor (LHR) expression by in vitro cultured mural granulosa cells.
  • Granulosa cells were isolated and cultured as described previously for varying lengths of time with fetal calf serum, +/- FSH. RNA was isolated as described and RT-PCR for LHR message was done as described using intron spanning primers.

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Abstract

La présente invention concerne une méthode permettant d'identifier un agent modifiant (inhibant, favorisant) l'activité du facteur 9 de différenciation de croissance (GDF-9). Ladite méthode consiste à combiner des cellules présentant un récepteur pour GDF-9 et un gène, l'expression du gène étant régulée par liaison du GDF-9 au récepteur; GDF-9; et un agent à identifier. La combinaison produite est maintenue dans des conditions appropriées à la liaison du GDF-9 aux récepteurs présents sur les cellules. On détermine ensuite le degré de liaison entre GDF-9 et les récepteurs présents sur les cellules, le degré de liaison du GDF-9 au récepteur, plus ou moins important selon la présence ou l'absence de l'agent à identifier, étant révélateur d'un agent modifiant l'activité de GDF-9.
PCT/US1999/007210 1998-04-01 1999-04-01 Analyse pour facteur 9 de differenciation de croissance WO1999050672A1 (fr)

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CA002325019A CA2325019A1 (fr) 1998-04-01 1999-04-01 Analyse pour facteur 9 de differenciation de croissance
NZ507484A NZ507484A (en) 1998-04-01 1999-04-01 In vitro assay for growth differentiation factor 9 based on the discovery that GDF-9 binds to granulosa cells found in mammalian ovaries
JP2000541529A JP2002510055A (ja) 1998-04-01 1999-04-01 成長分化因子９のアッセイ
MXPA00009653A MXPA00009653A (es) 1998-04-01 1999-04-01 Ensayo para el factor 9 de diferenciacion del crecimiento.
AU33777/99A AU753793B2 (en) 1998-04-01 1999-04-01 Assay for growth differentiation factor 9
EP99915200A EP1066528A1 (fr) 1998-04-01 1999-04-01 Analyse pour facteur 9 de differenciation de croissance

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007009166A1 (fr) * 2005-07-18 2007-01-25 Adelaide Research & Innovation Pty Ltd Modulation de l'apoptose des cellules de la granulosa
WO2007112386A3 (fr) * 2006-03-28 2008-05-02 Wyeth Corp Modulateurs de gdf-9/bmp-15 pour le traitement de troubles des os
CN110914686A (zh) * 2017-02-01 2020-03-24 新南创新私人有限公司 配子分泌的生长因子
CN113834936A (zh) * 2021-08-20 2021-12-24 李竞宇 生长分化因子9在预测胚胎发育潜力中的应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994015966A1 (fr) * 1993-01-12 1994-07-21 Johns Hopkins University School Of Medicine Facteur-9 de croissance et de differentiation

Patent Citations (1)

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Publication number Priority date Publication date Assignee Title
WO1994015966A1 (fr) * 1993-01-12 1994-07-21 Johns Hopkins University School Of Medicine Facteur-9 de croissance et de differentiation

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DONG J ET AL: "GROWTH DIFFERENTIATION FACTOR-9 IS REQUIRED DURING EARLY OVARIAN FOLLICULOGENESIS", NATURE, vol. 383, 10 October 1996 (1996-10-10), pages 531 - 535, XP002915645, ISSN: 0092-8674 *
ELVIN, JULIA A. ET AL: "Molecular characterization of the follicle defects in the growth differentiation factor 9 -deficient ovary", MOL. ENDOCRINOL. (1999), 13(6), 1018-1034, XP002114330 *
ELVIN, JULIA A. ET AL: "Paracrine actions of growth differentiation factor - 9 in the mammalian ovary", MOL. ENDOCRINOL. (1999), 13(6), 1035-1048, XP002114331 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007009166A1 (fr) * 2005-07-18 2007-01-25 Adelaide Research & Innovation Pty Ltd Modulation de l'apoptose des cellules de la granulosa
US8530235B2 (en) 2005-07-18 2013-09-10 Adelaide Research & Innovation Pty Ltd. Medium that supports culture of an oocyte and modulation of granulosa cell apoptosis
WO2007112386A3 (fr) * 2006-03-28 2008-05-02 Wyeth Corp Modulateurs de gdf-9/bmp-15 pour le traitement de troubles des os
US7790161B2 (en) 2006-03-28 2010-09-07 Wyeth Llc GDF-9/BMP-15 modulators for the treatment of bone disorders
CN110914686A (zh) * 2017-02-01 2020-03-24 新南创新私人有限公司 配子分泌的生长因子
CN113834936A (zh) * 2021-08-20 2021-12-24 李竞宇 生长分化因子9在预测胚胎发育潜力中的应用

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