WO2000066620A2 - Morphogen-induced enhancement of fertility - Google Patents

Abstract

The present invention provides methods and compositions for the treatment of human infertility, using the administration of morphogens to regulate ovarian follicle growth. The invention further provides methods and compositions to delay the onset or alleviate symptoms of menopause, and methods and compositions to decrease fertility.

Description

MORPHOGEN-INDUCED ENHANCEMENT OF FERTILITY

FIELD OF THE INVENTION

The invention relates generally to methods and compositions for the modulation of human fertility. More particularly, the present invention relates to methods and compositions the to enhance fertility, methods and compositions to delay the onset or alleviate symptoms of menopause, and methods and compositions to decrease fertility.

BACKGROUND OF THE INVENTION

Morphogens are members of the TGF-β superfamily that perform essential physiological functions in morphogenesis and organogenesis. Morphogens are expressed in a tissue-specific manner in many different cell types during embryonic and adult life in both vertebrates and invertebrates. The importance of morphogens in regulating crucial events in morphogenesis, organogenesis, and cytodifferentiation has been clearly established from studies of morphogen-deficient animals.

Morphogens, also referred to as osteogenic proteins (OPs) or bone morphogenic proteins (BMPs), are generally classified as a subgroup of the TGF-β superfamily of growth factors. Hogan, Genes & Development 10: 1580-1594 (1996). Members of the morphogen family of proteins include the mammalian osteogenic protein-1 (OP-1, also known as BMP-7, and the Drosophila homolog 60A), osteogenic protein-2 (OP-2, also known as BMP-8), osteogenic protein-3 (OP-3), BMP-2 (also known as BMP-2A or CBMP-2A, and the Drosophila homolog dpp), BMP-3, BMP-4 (also known as BMP-2B or CBMP-2B), BMP-5,

BMP-6 and its murine homolog Vgr-1, BMP-9, BMP- 10, BMP-11, BMP- 12, GDF3 (also known as Vgr2), GDF8, GDF9, GDF10, GDF11, GDF12, BMP- 13, BMP-14, BMP-15, GDF-5 (also known as CDMP-1 or MP52), GDF-6 (also known as CDMP-2), GDF-7 (also known as CDMP-3), the Xenopus homolog Vgl and NODAL, UNININ, SCREW, ADMP, and NEURAL.

Members of this family encode secreted polypeptides that share common structural features. The mature form of such proteins results from processing through a "pro-form" to yield a mature polypeptide chain competent to dimerize and containing a carboxyl terminal active domain of approximately 97-106 ammo acids. All members share a conserved pattern of cysteines in this domain and the active form of these proteins can be either a disulfide-bonded homodimer of a single family member or a heterodimer of two different members. See, e.g., Massague, Anna. Rev. Cell Biol. 6: 597 (1990); Sampath et al, J. Biυl. Chem. 265: 13198 (1990). See also, U.S. Patents 5,011 ,691, and 5,266,683; Ozkaynak et al,

EMBO J. 9: 2085-2093 (1990), Wharton et al, Proc. Natl. Acad. Sci USA 88: 92 14-9218 (1991); Ozkaynak, J. Biol. Chem. 267: 25220-25227 (1992); Celeste et al, Proc. Natl. Acad. Sci USA 87: 9843-9847 (1991); Lyons et al, Proc. Natl. Acad. Sci USA 86: 554-4558 (1989). These disclosures describe the amino acid and DNA sequences, as well as the chemical and physical characteristics, of morphogens. See also, Wozney et al, Science 242: 1528-1534

(1988); PCT application WO 93/00432; Padgett et al, Nature 325: 81-84 (1987); and Weeks, Cell 51: 861 -867 (1987).

The biological effects of morphogens are mediated by specific cell surface receptors, morphogen receptors exist as two subtypes, the type I receptors and the type II receptors. Both types of morphogen receptors are structurally similar and both types possess intrinsic serine/threonine kinase activity. Two type I morphogen receptors, BMPR-IA (or ALK-3) and BMPR-IB (or ALK-6), have been identified. One type II morphogen receptor, BMPR-II, has also been identified. Individually, either type I morphogen receptors or type II morphogen receptors can bind morphogen as a ligand with low affinity. However, both receptor types are necessary to achieve high affinity binding and ligand-mediated signal transduction. After the ligand-receptor complex is formed, the type II receptor phosphorylates and activates the type I receptor. The type I receptor then triggers downstream events in the morphogen-signaling pathway.

Although much is known about the cellular function and biological importance of morphogen signaling in a many embryonic and adult tissues, the role of morphogen signaling in the reproductive system is poorly understood. Thus far, no experimental evidence has shown that morphogen action is available in reproductive cells for any species. Messenger πbonucleic acids (mRNAs) encoding BMP-2, BMP-3, BMP-3b, BMP-6 and BMP- 15 have been identified in mammalian ovaries and expression of BMP-6 and BMP- 15 has been localized to the oocytes by in situ hybridization. However, nothing is known about the expression of receptors for morphogens or the functional consequences of morphogen receptor activation in the ovary.

Two hormones known to be important for the regulation of human fertility are the follicle stimulating hormone (FSH) and the luteinizing hormone (LH), each produced by the anterior hypothalmus in the brain. FSH stimulates ovarian follicle growth in females (a physiological process that depends on the hormone estrogen) and spermatogenesis in males. LH stimulates ovulation and luteinization of ovarian follicles in females (a physiological process that depends on the hormone progesterone) and testosterone secretion in males. Together, FSH and LH stimulate sex hormone release and regulate the hormonal balance of estrogen and progesterone. All four of these hormones are necessary for the development of the ovarian follicle, the female reproductive organ in which an oocyte (egg cell) is surrounded by one or more layers of granulosa cells, as well as other cells. In the final stage of ovarian differentiation, a cavity forms in the ovary follicle; the ovarian follicle is then termed a Graafian follicle. A major concept in ovarian physiology is that FSH-dependent ovarian follicle growth is marked by increasing synthesis of estrogen, but not progesterone, in the cells of the developing ovarian follicle. This transformation of the mature ovarian follicle and its theca interna into a corpus luteum after ovulation, and the formation of luteal tissue is termed luteinization. It has been known for many years that a "luteinization inhibitor" plays an important role in inhibiting follicular progesterone production, but the molecular nature of the "luteinization inhibitor" remains a mystery.

Thus, there is a need in the art for the identification of the long sought "luteinization inhibitor" in Graafian follicles during follicular growth and development and the medical use of the "luteinization inhibitor" in the treatment of human fertility.

SUMMARY OF THE INVENTION The invention provides a method for increasing fertility, by providing a "luteinization inhibitor" to a female subject in the form of a therapeuticaUy effective amount of a morphogen pharmaceutical. The morphogen is a peptide having an amino acid sequence selected from a sequence: (1) having at least 70% homology with the C-terminal seven-cysteine skeleton of human OP-1. amino acids 330-431 of SEQ ID NO: 2; (2) having greater than 60% amino acid sequence identity with said C-terminal seven-cysteine skeleton of human OP-1 ; (3) defined by SEQ ID NO: 5; (4) defined by SEQ ID NO: 6; (6) defined by SEQ ID NO: 7; (7) defined by SEQ ID NO: 8; or (8) defined by OPX, SEQ ID NO: 4. In one embodiment, the therapeuticaUy effective amount is nanomolar. The administration of the morphogen induces estrogen synthesis by the ovary of the subject, and can also attenuate progesterone synthesis by the ovary of the subject. The subject receiving the morphogen can have healthy ovary follicles, atretic ovary follicles, or both.

The invention also provides a method for alleviating symptoms of menopause or for delaying the onset of menopause, in which a therapeuticaUy effective amount of a morphogen is administered to the subject. The morphogen induces ovarian follicle growth, which is marked by increasing ovarian synthesis of estrogen The administration of the morphogen can also attenuate progesterone synthesis by the ovary of the subject. Morphogens can thus be used to prolong promote follicular growth over the duration of a women's life when she can have menstrual cycles. Because menstruation results from a balance of hormonal factors, those factors that promote follicular growth can be used to delay the onset of menstruation. The invention further provides a method for contraception. The method reduces ovarian follicular growth, and thus ovulation and production of hormones in the ovary, by administering to a subject a compound which interferes with the binding of the morphogen and its receptor on oocytes or ovarian granulosa cells .is administered. Treating a subject with the compound results in a subdued ovulation and an increased luteinization of the follicles, decreasing the fertility of the woman receiving the compound. In one embodiment, the compound that interferes the binding of the morphogen to its receptor is an anti-morphogen antibody. In another embodiment, the compound that interferes the binding of the morphogen to its receptor is an anti-receptor antibody. In yet another embodiment, the compound that interferes the binding of the morphogen to its receptor is a morphogen receptor antagonist. As used herein, the terms "morphogen," "bone morphogen," "osteogenic protein,"

"OP," "bone morphogenic protein," "BMP," "morphogenic protein" and "morphogenetic protein" all embrace the class of proteins typified by human osteogenic protein 1 (hOP-1). Nucleotide and amino acid sequences for hOP-1 are provided in SEQ ID NOS: 1 and 2, respectively. For ease of description, hOP-1 is considered a representative morphogen. It will be appreciated that OP-1 is merely representative of the TGF-β subclass of true tissue morphogens and is not intended to limit the description Other known and useful morphogens include, but are not limited to, BMP-2, BMP-3, BMP-3b. BMP-4, BMP-5, BMP-6, BMP-8, BMP-9, BMP-10, BMP-1 1 , BMP-12, BMP-13, BMP-15. GDF-1, GDF-2, GDF-3, GDF-5, GDF-6, GDF-7, GDF-8, GDF-9, GDF-10, GDF-1 1, GDF-12, 60A, NODAL, UNIVIN, SCREW, ADMP, and NEURAL, and morphogemcally-active ammo acid variants of any thereof

In specific embodiments, useful morphogens include those sharing the conserved seven cysteine skeleton, and sharing at least 70% ammo acid sequence homology (similarity), withm the C-termmal seven-cysteme skeleton of human OP-1, residues 330-431 of SEQ ID NO 2 (hereinafter referred to as the presently-preferred reference sequence) In another embodiment, the invention encompasses use of biologically active species (phylogenetic) variants of any of the morphogenic proteins recited herein, including conservative amino acid sequence variants, proteins encoded by degenerate nucleotide sequence vaπants, and morphogenically-active proteins sharing the conserved seven cysteine skeleton as defined herein and encoded by a DNA sequence competent to hybridize under standard stringency conditions to a DNA sequence encoding a morphogenic protein disclosed herein, including, without limitation, OP-1 or BMP-2 or BMP-4 Presently, however, the preferred reference sequence is that of residues 330-431 of SEQ ID NO 2 (OP-1)

In still another embodiment, morphogens useful m methods and compositions of the invention are defined as morphogemcally-active proteins having any one of the generic sequences defined herein, including OPX (SEQ ID NO 3) and Generic Sequences 7 and 8 (SEQ ID NOS 5 and 6, respectively), or Generic Sequences 9 and 10 (SEQ ID NOS 7 and 8, respectively) OPX encompasses the observed variation between the known phylogenetic counterparts of the osteogenic OP-1 and OP-2 proteins, and is described by the amino acid sequence presented herein below and in SEQ ID NO 3 Generic Sequence 9 is a 97 amino acid sequence containing the C-termmal six cysteine skeleton observed in hOP-1 (residues 335-431 of SEQ ID NO 2) and wherein the remaining residues encompass the observed \ aπation among OP-1, OP-2, OP-3, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-8, BMP-9, BMP-10, BMP-1 1, BMP-15, GDF-1. GDF-3, GDF-5, GDF-6, GDF-7, GDF-8, GDF-9, GDF-10, GDF-1 1, 60A, UNIVIN, NODAL, DORSALIN, NEURAL,

SCREW and ADMP That is, each of the non-cysteme residues is independently selected from the corresponding residue in this recited group of known, naturally-sourced proteins Generic Sequence 10 is a 102 ammo acid sequence which includes a five ammo acid sequence added to the N-terminus of the Generic Sequence 9 and defines the seven cysteine skeleton observed m hOP-1 (330-431 SEQ ID NO 2) Generic Sequences 7 and 8 are 97 and 102 ammo acid sequences, respectively, containing either the six cysteine skeleton (Generic Sequence 7) or the seven cysteine skeleton (Generic Sequence 8) defined by hOP-1 and wherein the remaining non-cysteme residues encompass the observed variation among OP-1, OP-2, OP-3, BMP-2, BMP-3, BMP-4, 60A, dpp, Vgl, BMP-5, BMP-6, Vgr-1, and GDF-1

Of particular interest herein are morphogens which, when provided to a specific tissue of a mammal, induce tissue-specific morphogenesis or maintain the normal state of differentiation and growth of that specific tissue In preferred demonstrative embodiments, the present morphogens induce the formation of vertebrate (e g , avian or mammalian) body tissues, such as but not limited to nerve, eye, bone, cartilage, bone marrow, ligament, tooth dentin, peπodontium, liver, kidney, lung, heart, or gastrointestinal lining The present demonstrations can be carried out in the context of developing embryonic tissue, or at an aseptic, unscarred wound site in post-embryonic tissue Methods of identifying such morphogens, or morphogen receptor agonists, are known in the art and include assays for compounds which induce morphogen-mediated responses (e g , induction of endochondral bone formation, induction of differentiation of metanephric mesenchyme, and the like) In a currently preferred demonstrative embodiment, morphogens of the present invention, when implanted m a mammal in conjunction with a matrix permissive of bone morphogenesis, are capable of inducing a developmental cascade of cellular and molecular events that culminates m endochondral bone formation See U S Patent 4,968,590 and Sampath, et at , Proc Natl Acad Sci USA 80 659 1-6595 (1983), the disclosures of which are incorporated herein by reference

BRIEF DESCRIPTION OF THE DRAWINGS

FIG 1 (panels 1-A through 1-M) are a tabular alignment of the ammo acid sequences of various naturally-occurring morphogens with a preferred reference sequence of human OP-1, residues 330-431 of SEQ ID NO 1 FIG 2 is a tabular presentation of alternative ammo acids for "Xaa" positions in generic sequences SEQ ID NOS 5, 6, and 9 that represent amino acid variations in known morphogens

FIG 3 is a tabular presentation of alternative ammo acids for "Xaa" positions in generic sequences SEQ ID NOS 5, 6, and 9 that represent ammo acid variations m known morphogens

FIG 4 is a tabular presentation of alternative ammo acids for "Xaa" positions in generic sequences SEQ ID NOS 7, 8, and 10 that represent ammo acid vaπations in known morphogens FIG 5 is a set of graphs showing the effects of morphogens on estrogen and progesterone production by granulosa cells Granulosa cells (5 x 10⁴ viable cells/well/200 μl) were cultured for 48 hours (hr) in serum-free medium containing androstenedione (1 μM), and either no additions (control), BMP-4 (3, 10, or 30 ng/ml), BMP-7 (3, 10, or 30 ng/ml), FSH (0 1, 0 3, 1, 3, or 10 ng/ml) or their combination After culture, estrogen levels (Panels A and C) and progesterone levels (Panels B and D) in the conditioned media were measured by radioimmunoassay

FIG 6 is a set of graphs showing the time course effect of BMP-7 on estrogen and progesterone production by granulosa cells Granulosa cells (5 x 10⁴ viable cells/well/200 μl) were cultured for 48 hr m serum-free medium containing androstenedione (1 μM), and FSH (3 ng/ml) m the absence or presence of BMP-7 (30 ng/ml) After culture, estrogen levels

(Panel A) and progesterone levels (Panel B) in the conditioned media were measured by radioimmunoassay

FIG 7 is a set of graphs showing that FSHβLuc expression is increased by morphogens and activm FIG 8 is a set of graphs showing dose-dependent inhibition of FSHβLuc expression using rabbit a i mOP-1 1

FIG 9 is a set of bar graphs showing the neutralizing effects of mOP-1 and activm antibodies DETAILED DESCRIPTION OF THE INVENTION

A. General

The invention provides methods for increasing fertility by inhibiting luteinization in the ovarian follicles Luteinization can be an important part of a healthy menstrual cycle when induced by the luteinization hormone (LH), an increase in the levels of which occur normally prior to ovulation However, luteinization is a complex differentiation process involving the interaction of extrinsic and mtraovaπan factors Undesirable luteinization caused by a low production of hormones by ovarian cells can result in a lowered fertility. In one embodiment, the invention provides a method for increasing fertility, by providing a "luteinization inhibitor" to a female subject in the form of a morphogen pharmaceutical

It is well known that an insufficiency in ovarian follicular growth can cause a decreased fertility Daly, Fertil Steal 51(1) 51-7 (1989) Reduced fertility can be due to an inappropriate luteinization, in which the luteal phase defect is associated with either an impaired follicular growth or an abnormal surge in LH levels Ayabe et al , Fertil Stenl 61(4) 652-6 (1994), Lewmthal et al , Fertil Stenl 46(5) 833-9 (1986)

The administration of morphogens can increase fertility by several mechanisms First, morphogens can act directly on the cells of the ovary to increase ovarian synthesis of estrogen Second, morphogens act on the pituitary to increase synthesis of FSH FSH then binds to granulosa cells in the ovaries, stimulating estrogen production High FSH levels also advance the preovulatory stage of the dominant follicle in the early follicular phase of the cycle

Furthermore, FSH controls follicle development in women at the recruitment-selection stage Messims et al , Hum Reprod 5(2) 153-6 (1990) Third, morphogens can act on ovarian cells to potentiate the effects of FSH Recent findings from the study of the regulation of follicular dex elopment show that the potentiating effect of vaπous growth factors on ovaπan sensitivity to FSH Lunenfeld et al , Bailheres Clin Obstet Gynaecol 4(3) 473-89 (1990) As shown below, administration of morphogens can act in a more direct manner to potentiate the effects of FSH Finally, morphogens can act on ovarian cells to increase synthesis of FSH

In one embodiment, the invention provides a method for alleviating symptoms menopause Menopause results from the ovaries decreasing their production of the sex hormones estrogen and progesterone The drop in estrogen levels causes the most common symptoms during menopause. Current methods for treating menopause include estrogen replacement therapies, taken in the form of oral tablets, skin patches, or injections. Estrogen circulates through the body to reduce the short-term changes of menopause. The combination therapy of estrogen plus progesterone is called hormone replacement therapy. As an alternative to estrogen replacement therapies or hormone replacement therapies, estrogen receptor modulators are used to treat menopause.

The invention provides a method of using morphogens to induce FSH-dependent ovarian follicle growth, which is marked by increasing ovarian synthesis of estrogen. The invention thus provides another treatment, which can be an alternative to or a supplement for estrogen replacement therapies. Moφhogens can be used to prolong promote follicular growth over the duration of a women's life when she can have menstrual cycles. Because menstruation results from a balance of hormonal factors, those factors that promote follicular growth can be used to delay the onset of menstruation. By promoting healthy follicular growth, the method of the invention also results in increased ovarian levels of estrogen. The invention also provides a method of contraception. Treating a female subject with a compound that prevents the binding of moφhogen to a moφhogen receptor on oocytes or ovarian granulosa cells will result m a subdued ovulation and an increased luteinization of the follicles. It is well known in the art that a woman's fertility is reduced as her ovulation diminishes, or when her ovaries' production of estrogen and other sex hormones decreases. The invention provides a method for reducing ovarian follicular growth and thus ovulation and production of hormones in the ovary, by administering compounds which interfere with moφhogen activity in promoting follicular growth early in the menstrual cycle. This decreases the fertility of the woman receiving the compounds that prevent the binding of moφhogen to BMP receptor. In the present invention, the cellular sites of expression of the moφhogen type IA, type IB, and type II receptors (BMPR-IA, BMPR-IB, BMPR-II) mRNAs and BMP-4 and BMP-7 mRNAs were characterized in the rat ovary, establishing for the first time the existence of a functional moφhogen ligand-receptor system in the ovary of any species. The genes encoding moφhogens and the genes encoding the famih of moφhogen receptors are expressed in a cell-type-specific manner. The co-expression of BMP-4 and BMP-7 mRNAs by the theca cells indicates the coordinate regulation of these two moφhogens during ovarian follicle development The expression of both moφhogens is stage-specific in the cycle of fol culogenesis, being very high in healthy follicles but barely detectable in follicles undergoing atresia This pattern of expression shows that the coordinate expression of these two moφhogens is subject to different patterns of regulation duπng follicle development and atresia

Tissue culture results demonstrate that moφhogens elicit key biological responses m granulosa cells Two types of responses were observed First, BMP-4 and BMP-7 both caused a time-dependent and dose-dependent amplification FSH-induced estrogen production and a time-dependent and dose-dependent attenuation of FSH-induced progesterone production The ED,₀ of the moφhogen responses (-10 ng/ml or ~3 x 10'°M) is equivalent to the reported Kd of the moφhogen receptor (2 54 x 10¹⁰M) This strongly suggests that moφhogen responses are mediated by cell surface receptors and are physiological Second, granulosa cells are more sensitive to FSH-action after moφhogen treatment This effect appears to be coupled only to estrogen production The interaction of nanomolar amounts of

BMP-4 and BMP-7 with moφhogen receptors causes marked stimulatory effects on FSH-mduced estiogen production and inhibitory effects on FSH-induced progesterone production, respectively Thus, moφhogens appear to influence FSH signaling pathways to promote estrogen production, and decrease progesterone production Growth hormone and msuhn- ke growth factor-I (IGF-I) are potent stimulators of

BMP-4 mRNA levels m human dental pulp fibroblasts cultured in vitro IGF-I is a potent stimulator of rat theca cell function Also, IGF-I expression is strong and weak m healthy and atretic follicles respectively Thus, IGF-I may be a physiological stimulus for moφhogens expression duπng ovarian follicle growth

B. Biochemical, Structural and Functional Properties of Bone Morphogenic

Proteins

In its mature, native form, natural-sourced moφhogen is a glycosylated dimer, typically having an apparent molecular weight of about 30-36 kDa as determined by SDS-PAGE When i educed, the 30 kDa protein giv es rise to tw o glycosylated peptide subunits having apparent molecular weights of about 16 kDa and 18 kDa In the reduced state, the protein has no detectable osteogenic activity The unglycosylated protein, which also has osteogenic activity, has an apparent molecular weight of about 27 kDa When reduced, the 27 kDa protein gives rise to two unglycosylated polypeptide chains, having molecular weights of about 14 kDa to 16 kDa Typically, the naturally-occurring morphogens are translated as a precursor, hav mg an N-termmal signal peptide sequence typically less than about 30 residues, followed by a "pro" domain that is cleaved to yield the mature C-termmal domain The signal peptide is cleaved rapidly upon translation, at a cleavage site that can be predicted m a given sequence using the method of Von Heijne, Nucleic Acids Res 14 4683-4691 (1986) The pro domain typically is about three times larger than the fully processed mature C-terminal domain

Moφhogens useful herein include any known naturally-occurπng nativ e proteins including allelic, phylogenetic counteφart and other variants thereof, whether naturally-occurring or biosynthetically produced (e g , including "muteins" or "mutant proteins"), as well as new, osteogemcally active members of the general moφhogemc family of proteins

Particularly useful sequences include those comprising the C-termmal 97 or 102 amino acid sequences of dpp (from Drosophila), Vgl (from Xenopus), Vgr-1 (from mouse), the OP-1 and OP-2 proteins (see U S Patents 5,011 ,691 and 5,266,683, Ozkaynak et al , EMBO J 9 2085-2093 (1990)), as well as the proteins referred to as BMP-2, BMP-3, BMP-4 (see WO 88/00205, U S Patent 5,013,649 and WO 91/18098), BMP-5 and BMP-6 (see

WO 90/11366, PCT/U590/01630), BMP-8 and BMP-9 Other proteins useful in the practice of the invention include active forms of OP-1, OP-2, OP-3, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-9, GDF-5, GDF-6, GDF-7, dpp, Vgl, Vgr, 60A protein, GDF-1, GDF-3, GDF-5, GDF-6, GDF-7, BMP-10, BMP-11, BMP-13, BMP-15, UNIVIN, NODAL, SCREW, ADMP or NURAL and ammo acid sequence variants thereof In one currently preferred embodiment, moφhogens include any one of OP-1 , OP-2, OP-3, BMP-2, BMP-4, BMP-5, BMP-6, BMP-9, and ammo acid sequence variants and homologs thereof, including species homologs, thereof Publications disclosing OP-1 and OP-2 sequences, as well as their chemical and physical properties, include U S Patents 5,01 1 ,691 and 5,266,683, incoφorated by reference herein In preferred embodiments, moφhogens for use in methods of the invention include proteins having at least 70% homology with the amino acid sequence of the C-terminal seven-cysteine skeleton of human OP-1, SEQ ID NO: 2, and having the ability to induce endochondral bone formation in the Reddi and Sampath assay described herein. Compounds that meet these requirements are considered functionally equivalent to a known response moφhogen. To determine whether a candidate amino acid sequence is functionally equivalent to a reference moφhogen, the candidate sequence and the reference sequence are aligned. The first step for performing an alignment is to use an alignment tool, such as the dynamic programming algorithm described in Needleman et al., J. Mol. Biol. 48: 443 (1970), and the Align Program, a commercial software package produced by DNAstar, Inc. the teachings of which are incoφorated by reference herein. After the initial alignment is made, it is then refined by comparison to a multiple sequence alignment of a family of related proteins, such as those shown in FIG. 1A through 1M, which is a multiple sequence alignment of a family of known moφhogens, including hOP-1. Once the alignment between the candidate and reference sequences is made and refined, a percent homology score is calculated. The individual amino acids of each sequence are compared sequentially according to their similarity to each other.

Similarity factors include similar size, shape and electrical charge. One particularly preferred method of determining amino acid similarities is the PAM25O matrix described in Dayhoff et al., 5 ATLAS OF PROTEIN SEQUENCE AND STRUCTURE 345-352 (1978 & Supp.), incoφorated by reference herein. A similarity score is first calculated as the sum of the aligned pairwise amino acid similarity scores. Insertions and deletions are ignored for the puφoses of percent homology and identity. Accordingly, gap penalties are not used in this calculation. The raw score is then normalized by dividing it by the geometric mean of the scores of the candidate compound and the seven cysteine skeleton of hOP-1. The geometric mean is the square root of the product of these scores. The normalized raw score is the percent homology.

In an alternative preferred embodiment, a functionally-equivalent moφhogen sequence shares at least 60% amino acid identity with a reference sequence. That is, any 60% of the aligned amino acids are identical to the corresponding amino acids in the reference sequence.

Any one or more of the naturally-occurring or biosynthetic moφhogens disclosed herein may be used as a reference sequence to determine whether a candidate sequence falls within the moφhogen family. In a preferred embodiment, the reference sequence is the C-terminal seven-cysteine skeleton sequence of human OP-1 as shown in SEQ ID NO: 2. Examples of conservative substitutions for use in the above calculations include the substitution of one amino acid for another with similar characteristics, e.g., substitutions within the following groups are well-known: (a) valine, glycine; (b) glycine, alanine; (c) valine, isoleucine, leucine; (d) aspartic acid, glutamic acid; (e) asparagine, glutamine; (f) seine, threonine; (g) lysine, arginine, methionine; and (h) phenylalanine, tyrosine. The term "conservative variant" or conservative variation" also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid in a given polypeptide chain, provided that antibodies having binding specificity for the resulting substituted polypeptide chain also have binding specificity (i.e., "crossreact" or "immunoreact" with) the unsubstituted or parent polypeptide.

In a preferred embodiment, moφhogens useful in the resent invention are defined by a generic amino acid sequence that represents variations in known moφhogens. For example, SEQ ID NOS: 4 and 5 encompass observed variations between preferred moφhogens, including OP-1, OP-2, OP-3, CBMP-2A, CBMP-2B, BMP-3, 60A, dpp, Vgl, BMP-5, BMP-6, Vgr-1, and GDF-1. SEQ ID NO: 5 includes all of SEQ ID NO: 4, and also includes at its N-terminus the five amino acid sequence of SEQ ID NO: 8. The generic sequences include both the amino acid identity shared by these sequences in the C-terminal domain, defined by the six- and seven-cysteine skeletons (SEQ ID NOS: 5 and 6, respectively), and alternative amino acids for variable positions within the sequence. Positions that allow for alternative amino acids are represented by "Xaa". FIG. 2 shows the alternative amino acids for each "Xaa" position in SEQ ID NOS: 5, 6 and 9. For example, referring to SEQ ID NO: 6 and FIG. 2, the "Xaa" at position 2 may be a tyrosine or a lysine. The generic sequences provide an appropriate cysteine skeleton for inter- or intramolecular disulfide bonding, and contain certain critical amino acids likely to influence the tertiary structure of the proteins. In addition, the "Xaa" at position 36 in SEQ ID NO: 5, or at position 41 in SEQ ID NO: 6, may be an additional cysteine, thereby encompassing the moφhogenically-active sequences of OP-2 and OP-3. In another embodiment, useful moφhogens include those defined by SEQ ID NOS: 7 or 7, which are composite amino acid sequences of the following moφhogens: human OP-1, human OP-2, human OP-3, human BMP-2, human BMP-3, human BMP-4, human BMP-5, human BMP-6, human BMP-8, human BMP-9, human BMP-10, human BMP-11, Drosophila 60A, Xenopus Vg-1, sea urchin UNIVIN, human CDMP-1 (mouse GDF-5), human CDMP-2 (mouse GDF-6, human BMP-13), human CDMP-3 (mouse GDF-7, human BMP- 12), mouse GDF-3, human GDF-1, mouse GDF-1 , chicken DORSALIN, Drosophila dpp, Drosophila

SCREW, mouse NODAL, mouse GDF-8, human GDF-8, mouse GDF-9, mouse GDF-10, human GDF-1 1 , mouse GDF-1 1, human BMP-15, and rat BMP-3b. SEQ ID NO: 8 includes all of SEQ ID NO: 7 and also includes at its N-terminus the five amino acid sequence of SEQ ID NO: 10. SEQ ID NO: 7 accommodates the C-terminal six-cysteine skeleton, and SEQ ID NO: 8 accommodates the seven-cysteine skeleton. Positions that allow for alternative amino acids are represented by "Xaa". FIG. 4 shows the alternative amino acids for each "Xaa" position in SEQ ID NOS: 7, 8 and 10.

As noted above, certain preferred moφhogen sequences useful in this invention have greater than 60% identity, preferably greater than 65% identity, with the amino acid sequence defining the preferred reference sequence of hOP-1. These particularly preferred sequences include allelic and phylogenetic variants of the OP-1 and OP-2 proteins, including the Drosophila 60A protein, as well as the closely related proteins BMP-5, BMP-6 and Vgr-1. Accordingly, in certain particularly preferred embodiments, useful moφhogens include proteins comprising the generic amino acid sequence SEQ ID NO: 3 (referred to herein as "OPX"), which defines the seven-cysteine skeleton and accommodates the homologies between several identified variants of OP-1 and OP-2. Positions that allow for alternative amino acids are represented by "Xaa". FIG. 4 shows the alternative amino acids for each "Xaa" position in SEQ ID NO: 3.

In still another preferred embodiment, useful moφhogens include those having an amino acid sequence encoded by a polynucleotide that hybridizes under high stringency conditions with DNA or RNA encoding a reference moφhogen. Standard stringency conditions are well characterized in standard molecular biology texts. See generally, MOLECULAR CLONING: A LABORATORY MANUAL, (Sambrook et al, eds., 1989); DNA CLONING, Vol. I & II (D.N. Glover ed., 1985); OLIGONUCLEOTIDE SYNTHESIS (M.J. Gait ed., 1984); NUCLEIC ACID HYBRIDIZATION (B. D. Hames and S.J. Higgins eds., 1984); B. Perbal, A

PRACTICAL GUIDE TO MOLECULAR CLONING (1984). In another embodiment, moφhogens useful m the invention include the soluble complex form comprising a mature moφhogen dimer linked to a moφhogen pro domain or a solubihty-enhancmg fragment thereof A solubility-enhancing fragment is any N-termmal or C-termmal fragment of a moφhogen pro domain that forms a complex with the mature moφhogen dimer and increases the solubility of the moφhogen dimer Preferably, the soluble complex comprises a moφhogen dimer and two pro domain peptides Moφhogen soluble complex is described in published application WO 94/03600, mcoφorated by reference herein

In yet another embodiment, useful moφhogens include biologically active biosynthetic constructs, including novel biosynthetic moφhogens and chimeπc proteins designed using sequences from two or more known moφhogens See U S Patent 5,011,691, incoφorated by reference herein (e g , COP-1, COP-3, COP-4, COP-5, COP-7, and COP-16)

C. Formulations and Methods of Treatment

Compositions of the present invention (i e , comprising a molecules capable of releasing moφhogen inhibition administered, alone or in combination with a moφhogen) may be administered by any route which is compatible with the particular molecules and, when included, with the particular moφhogen Thus, as appropriate, administration may be oral or parenteral, including intravenous and lntrapeπtoneal routes of administration. In addition, administration may be by periodic injections of a bolus of the composition, or may be made more continuous by intravenous or mtrapeπtoneal administration from a reservoir which is external (e g , an I v bag) or internal (e g , a bioerodable implant, or a colony of implanted, moφhogen-producmg cells)

Therapeutic compositions of the present invention may be provided to an individual by am suitable means, directly (e g , locally, as by injection, implantation or topical administration to a tissue locus) or systemically (e g , parenteraUy or orally) Where the composition is to be provided parenteraUy, such as by intravenous, subcutaneous, intramolecular, ophthalmic, mtrapeπtoneal, intramuscular, buccal, rectal, vaginal, mtraorbital, intracerebral. mtracramal, intraspmal, mtraventπcular, mtrathecal, mtraci sternal, mtracapsular, mtranasal or by aerosol administration, the composition preferably comprises part of an aqueous or physiologically compatible fluid suspension or solution Thus, the carrier or \ ehicle is phvsiologically acceptable so that in addition to delivery of the desired composition to the patient, it does not otherwise adversely affect the patient's electrolyte and/or volume balance The fluid medium for the agent thus can comprise nonrial physiologic saline (e g 9 85% aqueous NaCl, 0 15 M, pH 7-7 4)

For moφhogens, association of the mature moφhogen dimer with a moφhogen pro domain results in the pro form of the moφhogen which typically is more soluble m physiological solutions than the corresponding mature form In fact, endogenous moφhogens are thought to be transported (e g , secreted and circulated) in the mammalian body in this form This soluble form of the protein can be obtained from culture medium of moφhogen-secretmg mammalian cells, e g cells transfected with nucleic acid encoding and competent to express the moφhogen Alternatively, a soluble species can be formulated by complexing the mature, moφhogemcally-active polypeptide dimer (or an active fragment thereof) with a moφhogen pro domain polypeptide or a solubility-enhancing fragment thereof Solubility-enhancing pro domain fragments can be any N-terminal, C-termmal or internal fragment of the pro region of a member of the moφhogen family that complexes with the mature polypeptide dimer to enhance stability and/or dissolubility of the resulting noncovalent or convalent complex Typically, useful fragments are those cleaved at the proteolytic site Mg-Xaa-Xaa-Mg A detailed description of soluble complex forms of moφhogenic proteins, including how to make, test and use them, is described in WO 94/03600 In the case of OP-1, useful pro domain polypeptide fragments include the mtact pro domain polypeptide (residues 30-292) and fragments 48-292 and 158-292, all of SEQ ID NO 2 Another molecule capable of enhancing solubility and particularly useful for oral administrations, is casein For example, addition of 0 2% casein increases solubility of the mature active form of OP-1 by 80% Other components found in milk and/or various serum piotems may also be useful

Useful solutions for parenteral administration may be prepared by any of the methods w ell known m the pharmaceutical art, described, for example, in REMINGTON'S

PHARMACEUTICAL SCIENCES (Gennaro, A , ed ), Mack Pub , 1990 Formulations of the therapeutic agents of the invention may include, for example, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes, and the like Formulations for direct administration, in particular, may include glycerol and other compositions of high viscosity to help maintain the agent at the desired locus Biocompatible, preferably bioresorbable, polymers, including, for example, hyaluromc acid, collagen, tπcalcium phosphate, polybutyrate, lactide, and glycohde polymers and lactide/glycohde copolymers, may be useful excipients to control the release of the agent in vivo Other potentially useful parenteral delivery systems for these agents include ethylene-vmyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and hposomes Formulations for inhalation administration contain as excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or oily solutions for administration in the form of nasal drops, or as a gel to be applied intranasally Formulations for parenteral administration may also include glycocholate for buccal administration, methoxysa cylate for rectal administration, or cutπc acid for vaginal administration Suppositories for rectal administration may also be prepared by mixing the molecule capable of releasing moφhogen inhibition (alone or in combination with a moφhogen) with a non-irritating excipient such as cocoa butter or other compositions which are solid at room temperature and liquid at body temperatures

Formulations for topical administration to the skm surface may be prepared by dispersing the molecule capable of releasing moφhogen inhibition (alone or in combination with a moφhogen) with a dermatologically acceptable carrier such as a lotion, cream, ointment or soap Particularly useful are carriers capable of forming a film or layer over the skm to localize application and inhibit removal For topical, administration to internal tissue surfaces, the agent may be dispersed in a liquid tissue adhesive or other substance known to enhance adsoφtion to a tissue surface For example, hydroxypropylcellulose or fibπnogen/thrombin solutions may be used to advantage Alternatively, tissue-coating solutions, such as pectm-containmg formulations may be used

Where the composition is intended for use as a therapeutic for disorders of the CNS, an additional problem must be addressed overcoming the blood-bram barner, the bram capillary wail structure that effectively screens out all but selected categories of substances present in the blood, preventing their passage into the bram The blood-bram barrier can be bypassed effectively by direct infusion of the molecule capable of releasing moφhogen inhibition (alone or in combination with a moφhogen) into the bram, or by mtranasal administration or inhalation of formulations suitable for uptake and retrograde transport by olfactory neurons D. Bioassav of Osteogenic Activitv : Endochondral Bone Formation and Related Properties

The art-iecognized bioassay for bone induction described by Sampath and Reddi, Pioc Natl Acad Sci USA 80 659 1-6595 (1983) and U S Patent 4,968,590), the disclosures of which are incoφorated by reference herein, are useful to establish the efficacy of a given device or formulation Briefly, the assay consists of depositing test samples in subcutaneous sites in recipient rats under ether anesthesia A vertical incision (1 cm) is made under steπle conditions in the skm over the thoracic region, and a pocket is prepared by blunt dissection In certain circumstances, approximately 25 mg of the test sample is implanted deep into the pocket and the incision is closed with a metallic skm clip The heterotropic site allows for the study of bone induction without the possible ambiguities resulting from the use of orthotopic sites

The sequential cellular reactions occurπng at the heterotropic site are complex The multi-step cascade of endochondral bone formation includes binding of fibπn and fibronectm to implanted matrix, chemotaxis of cells, proliferation of fibroblasts, differentiation into chondroblasts, cartilage formation, vascular invasion, bone formation, remodeling, and bone marrow differentiation

Successful implants exhibit a controlled progression through the stages of protem-mduced endochondral bone development including (1) transient infiltration by polymoφhonuclear leukocytes on about day one, (2) mesenchymal cell migration and proliferation on about days two and three, (3) chondrocyte appearance on about days five and six, (4) cartilage matrix formation on about day seven, (5) cartilage calcification on about day eight, (6) vascular invasion, appearance of osteoblasts, and formation of a new bone on about days nine and ten, (7) appearance of osteoblastic and bone remodeling on about days twelve to eighteen, and (8) hematopoietic bone marrow differentiation in the ossicle on about day twenty-one The time course of this process varies according to the matrix

Histological sectioning and staining is preferred to determine the extent of osteogenesis in the implants Staining with toluidine blue or hemotoxy n/eosin clearly demonstrates the ultimate dev elopment of endochondral bone Twelve day bioassays are sufficient to determine whether bone inducing activity is associated with the test sample Additionally, alkaline phosphatase activity can be used as a marker for osteogenesis. The enzyme activity can be determined spectrophotometrically after homogenization of the excised test material. The activity peaks at 9-10 days in vivo and thereafter slowly declines. Samples showing no bone development by histology should have no alkaline phosphatase activity under these assay conditions. The assay is useful for quantitation and obtaining an estimate of bone formation very quickly after the test samples are removed from the rat. For example, samples containing moφhogen at several levels of purity have been tested to determine the most effective dose/purity level, in order to seek a formulation that could be produced on an industrial scale. The results as measured by alkaline phosphatase activity level and histological evaluation can be represented as "bone forming units". One bone- forming unit represents the amount of protein that is needed for half maximal bone forming activity on day 12. Additionally, dose curves can be constructed for bone inducing activity in vivo at each step of a purification scheme by assaying various concentrations of protein. Accordingly, the skilled artisan can construct representative dose curves using only routine experimentation.

Example 1 A Functionally Active Morphogen System in the Ovary

1.1 In situ hybridization of BMP-4 and BMP-7, and type IA, type IB, and type II morphogen receptors in adult rat ovaries

In situ hybridization was used to determine the localization and level of expression of moφhogens and moφhogen receptors in adult rat ovaries. Probes to the moφhogens, BMP-4 and BMP-7, and probes to the type IA, type IB, and type II moφhogen receptors were used.

Reagents and supplies

The recombinant proteins, Xenopus BMP-4, human BMP-7, and human activin-A, were prepared as previously described by Kubo et al, Biol. Reprod. 58: 712-718 (1998). Ovine FSH (NIDDK-oFSH-S 1 , 4453 IU/mg) was supplied by the National Hormone and Pituitary Program of the NIDDK (Rockville, MD). McCoy's 5a medium, Medium 199, and dinucleotide triphosphates were purchased from Gibco BRL (Grand Island, NY). Cell culture plates were purchased from Falcon (Lincoln Park, NJ). Reagents for RT-PCR were obtained from Perkin Elmer (Foster City, CA). Cell culture

Twenty-three day old Sprague-Dawley rats (Harlan Industries, Indianapolis, IN) were implanted with silastic capsules containing 10 mg of diethylstilbestrol (DES) to increase granulosa cell number. Ovaries were removed and the granulosa cells isolated and cultured as previously described by Erickson and Hsueh, Endocrinology 102, 1275-1282 (1978).

Granulosa cells (5 x 10⁴ viable cells) were pipetted into 96-well culture plates containing 200 : 1 (final volume) of tissue culture medium (McCoy's 5a Medium containing 100 U/ml penicillin, 100 mg/ml streptomycin sulfate, 2 mM L-glutamine, and 1 μM androstenedione). Granulosa cells were cultured for up to 48 hr at 37^°C in water-saturated atmosphere containing 5% CO₂ in air with the indicated concentrations of FSH, BMP-4, BMP-7, or activin-A. After culture, the levels of progesterone and estrogen in the media were measured by radioimmunoassay as previously described by Wang et al., J Biol Chem 254, 11330-11336 (1979).

Construction of probe plasmids Total RNA from 27-day old rat ovaries was prepared. Single-stranded cDNA was synthesized by reverse transcriptase and then subjected to PCR as described previously by Shimasaki et al, J Biol Chem 266, 10646-10653 (1991). To design primers for PCR, DNA sequences of rat BMP-4 and BMPR-IA were obtained from GenBank. PCR primers for rat BMP-7, BMPR-IB, and BMPR-II were designed by choosing the homologous DNA sequence regions between human and mouse homologues of BMP-7, BMPR-IB and BMPR-II cDNAs which were available from GenBank. Specifically, these primers are derived from the cDNA clones at nucleotides 737-757 and 1 181-1200 (accession number of the cDNA clone is Z22607) for BMP-4 (Chen et al, Biochem. Biophys. Ada 1174, 289-292 (1993)); nucleotides 497-514 and 865-882 (accession number X56906) for BMP-7 (Ozkaynak et al, Biochem Biophys Res Commun 179, 116-123 (1991)); nucleotides 441-460 and 876-895 (accession number D38082) for BMPR-IA (Takeda et al, Biochem Biophys Res Commun 204, 203-209 (1994)); nucleotides 528-547 and 965-984 (accession number U89326) for BMPR-ffi; and nucleotides 525-544 and 895-904 (accession number AF003942) for BMPR-II. These primers were selected from different exons of the corresponding genes to discriminate PCR products that might arise from possible chromosome DNA contaminants. PCR was performed under the following conditions: 35 cycles, annealing at 50^°C for 30 sec; extension at 72^°C for 30 sec; denaturation at 94 C for 30 sec All PCR products were cloned into pBluescπpt SK+ plasmid and their DNA sequences confirmed

In situ hybridization

In situ hybridizations were performed as previously described by Nakatam et al Encloci otology 129, 1521-1529 (1991 ), with minor modifications Eight consecutiv e sections

(8 μm) were cut from each ovary and mounted onto poly-L lysine coated glass slides The sections were digested with protemase K, acetylated, washed and dehydrated Each antisense and sense cRNA probe was prepared by means of in vitro transcπption using T3 or T7 RNA polymerase Hybridization was carried out with the ^3:,S-labeled RNA probe (4-6 x 10⁶ cpm/ml) in a solution containing 50%> (vol/vol) deiomzed formamide, 0 3 M NaCl, 10 mM

Tns (pH 8 2), 1 mM EDTA, 0 05% yeast tRNA, 10 mM dithiothreitol, 1 x Denhardt's solution and 10%o dextran sulfate Hybridization solution (20 μl) was placed over each section and covered with a 60 x 22 mm acid washed, sihconized covershp Covershps were sealed with liquid DPX Sections were hybridized for 16 hr at 58-60°C in a humidified chamber After hybridization, the sections were treated with πbonuclease A and washed in 15 mM

NaCl/1 5 mM sodium citrate at 60-62°C for 30 mm Dehydrated slides were exposed to X-ray film for several days After adequate X-ray film images were obtained, the ovary sections were treated with xylene, rmsed m 100% ethanol, air dried, and then coated with Kodak NTB-2 liquid autoradiograph emulsion Slides were exposed for four weeks at 4°C in a desiccated dark box After exposure, the slides were developed (Kodak D19, 3 5 mm, 14°C), rinsed bπefly in distilled water and fixed After washing in distilled water for 1 hr, slides were lightly counterstamed with hematoxylm and eosin After an autoradiography and counterstammg, the sections were analyzed microscopically The in situ hybridizations were performed at least two times for each moφhogen ligand and receptor using one ovary from six different animals in each experiment

Results

The mRNAs foi BMP-4, BMP-7, and the BMPR-IA, BMPR-IB, and BMPR-II receptors weie expiessed in a tissue-specific manner in the adult lat ovary BMP-7 mRNA was present m the theca interstitial cells of healthy Graafian (dominant) follicles, but was undetectable in other ovarv cell types Hybridization with the control sense BMP-7 cRNA probe showed a nonspecific background signal, this was true for the other control sense probes used m these experiments BMP-4 mRNA was also expressed strongly in the theca cells of the dominant Graafian follicles, being present in both the theca interstitial and theca externa cells A weak but variable BMP-4 signal was observed in some coφora lutea and surface epithelial cells BMP-4 mRNA was not detectable in the other ovarian cell types

The mRNAs for BMPR-IA and BMPR-IB are widely expressed in the rat ovary, with the strongest hybridization signals being observed in the granulosa cells and oocytes of developing follicles The intensity of the signals for BMPR-IB were higher than those for BMPR-IA Hybridization signals for BMPR-II were most intense m the granulosa cells of all growing follicles (healthy and atretic) after the secondary stage The BMPR-II message was weakly expiessed m some coφora lutea A weak BMPR-II signal was observed in growing oocytes of primary follicles (those with a single layer of cuboidal granulosa cells), but none was observed in oocytes m late pre-antral and Graafian follicles No BMPR-II signal above background was observed m the other ovary cell types Conclusions

Moφhogens are expressed strongly in the ovary, being prominent in thecal cells High levels of moφhogen receptor expression found in granulosa cells Moφhogen receptor (BMPR-IA, BMPR-IB, and BMPR-II) mRNAs are uniformly expressed at high levels m all granulosa cells m all follicles, healthy as well as atretic, suggesting that the granulosa cells are important targets for moφhogen signaling These results suggest a paracπne role for moφhogens in regulating ovarian follicle growth

1.2 Effects of BMP-4 and BMP-7 on basal and FSH-stimulated estrogen and progesterone production by granulosa cells.

These observations obtained m Example 1 1 suggest a paracπne role for moφhogens whereby moφhogens produced by thecal cells interact with moφhogen receptors in the granulosa cells to regulate biological responses by a paracπne mechanism To assess this potential paracπne lole, the effects of two moφhogens, BMP-4 and BMP-7, were assessed on basal and FSH-stimulated estrogen and progesterone production in primary cultures of rat granulosa cells grown in serum-free medium

?^*? When granulosa cells were cultured for 48 hr in the absence of FSH, there was no detectable estrogen or progesterone in the medium. As illustrated in FIG. 5, treatment with BMP-4 or BMP-7 did not significantly affect these baseline levels.

FSH markedly increased estrogen and progesterone production in a dose dependent manner (ED₅₀ for FSH stimulated E= 0.52 ± 0.10; P= 1.19 ± 0.13). As illustrated in FIG. 5 A and C, BMP-4 and BMP-7 significantly modified the levels of FSH-stimulated estrogen and progesterone production. The levels of FSH-induced estrogen production were significantly increased (~2 to 3-fold) by both moφhogens and the effects were dose-dependent (ED₅₀ for BMP-4 = 89 ± 0.4 ng/ml; ED₅₀ for BMP-7 = 1 1.0 ± 1.0 ng/ml). Besides increasing the magnitude of the estrogen levels, BMP-4 and BMP-7 also caused a significant increase

(-1.5-fold; p<0.05) in FSH sensitivity with respect to estrogen production. In contrast to the positive effects on estrogen production, both BMP-4 and BMP-7 caused marked decreases (approximately 60%) in FSH-induced progesterone production. See FIG. 5B and D. These effects on FSH-induced progesterone production were dose dependent (ED₅₀ for BMP-4 = 10.9 ± 1.5 ng/ml; ED₅₀ for BMP-7 = 1 1.6 ± 3.2 ng/ml).

1J Time course of BMP-7 effects on FSH-stimulated estrogen and progesterone production by granulosa cells

In order to assess the time course of the effects of moφhogens on FSH-induced estrogen and progesterone production, granulosa cells were incubated in the presence of BMP-7 and FSH for a period of 72 hr and the levels of estrogen and progesterone were determined every 12 hr.

After a 24 hr lag phase, FSH induced a progressive increase in estrogen production which reached high levels after 72 hr of incubation. The 24 hr lag phase reflects the time needed for FSH to induce P450_AROMATASE activity. Richards, Endocr. Rev 15, 725-751 (1994). As depicted in FIG. 6, co-treatment of FSH with a saturating dose of BMP-7 (30 ng/ml) further increased the levels of estrogen (~2-fold) at each time point, but produced no change in the rate of FSH-induced estrogen accumulation throughout the 72 hr. By contrast, BMP-7 decreased the rate of FSH-stimulated progesterone accumulation by approximately 12 hr. The data in FIG. 6 illustrate that BMP-7 completely inhibited the FSH stimulation of progesterone at 24 hr and then continued to suppress progesterone levels (>50%) at 48 and 72 hr of culture. These findings suggest that BMP-7 suppresses both the maximal and relative rates of FSH-induced progesterone production.

Conclusions

The observation that BMPR-IA, BMPR-IB, and BMPR-II expression is strongest in the granulosa cells suggests that these cells are important targets for moφhogen signaling.

Moφhogen receptor mRNAs are uniformly expressed at high levels in all granulosa cells in all follicles, healthy as well as atretic. These results implicate moφhogens in normal follicle development and in the events leading to follicle death by apoptosis.

It is well established that the mechanism by which FSH stimulates estrogen and progesterone production involves the induction of the expression of specific steroidogenic genes. Evidence that SMAD proteins mediate moφhogen signaling and cyclic AMP mediates FSH signaling suggests the differential regulation might cross-talk between these different signaling pathways. A possible mechanism for moφhogen enhancement of estrogen production can be that the FSH signaling pathway receives positive regulatory inputs through SMAD proteins that lead to increases in P450_AROMATASE activity. A possible mechanism for moφhogen inhibition of progesterone production can be that SMAD proteins are negative regulator for the FSH signals that induce enzymes in the progesterone biosynthetic pathway, including StAR (steroidogenic acute regulatory protein), P450_scc (side-chain cleavage) or β-hydroxysteroid dehydrogenase.

Example 2 Morphogen Enhancement of FSH Expression in Pituitary

2.1 Stimulation of FSHβLUC activity by morphogen and activin

Pituitary cells from transgenic mice harboring FSHβLUC were dispersed and cultured for two days before treatments. The FSHβLUC construct contains an ovine FSHβ promoter driving a luciferase gene and functions in several transgenic mouse lines. Luciferase was expressed only in the pituitary and was regulated as if it were FSHβ itself. Therefore, luciferase activity seems to reflect normal FSH expression.

Cells were pretreated with follistatin (follicle stimulating hormone suppressing protein; FSP) (250 ng/ml). Follistatin suppresses the release of by FSH and LH. Follistatin was withdrawn and OP-1 , BMP-6, or activm- A was added Four hours after addition of a moφhogen or activin-A, the cells were harvested and assayed for luciferase activitv The data are representative of >3 individual experiments As illustrated m FIG 7, OP-1, BMP-6, and activm-A enhanced FSH expression in these mouse pituitary cell lines The moφhogen effect on FSH expression was dose-dependent Cultured pituitary cells from FSHβLUC transgenic mice were treated with rabbit antι-mOP-1 (0 1 to 10 μl/ml) or follistatin (250 ng/ml) for 24 hr After a 24 hr incubation, luciferase activity was measured As depicted in FIG 8, the antι-mOP-1 antibody produced a dose-dependent decrease in FSH expression, as measured by luciferase activity

2.2 FSHβLUC activity is inhibited by anti-morphogen antibodies but not by anti-activin antibodies

In order to assess the specificity of the enhanced FSH expression by a moφhogen and activin observed Example 2 1 , the cultured pituitary cells from FSHβLuc transgenic mice were treated for 24 hr with antibodies to mOP- 1 , activin- A, or activin-B and then assayed for luciferase activity

As depicted in FIG 9, the rabbit and sheep antι-mOP-1 antibodies were both capable of blocking the FSH expression in mouse pituitary cultures However, neither anti-activin-A antisera nor anti-activin-B antisera significantly inhibit FSHβLUC in the mouse pituitary system FSHβLUC activity was mildly blocked by monoclonal antibody IB 12 at 100 μg/ml (weak inhibition) but not by the 12G3 monoclonal antibody

These results suggest that moφhogens are the primary drive of FSH synthesis in the pituitary Further, because antι-OP-1 blocks FSHβLUC expression almost as well as follistatin, follistatin may inhibit FSH expression by binding and inactivating moφhogens or moφhogen receptors

Example 3 Morphogen Regulation of Follicle-Stimulating Hormone

Follicle-stimulating hormone (FSH) is produced m pituitary gonadotropes as an α/β heterodimer, and synthesis of the β subunit is the rate-limiting step in overall FSH production Synthesis of FSHβ is regulated by activm and inhibin, both of which are members of the transforming growth factor β (TGFβ) superfamily Bone moφhogenetic proteins (BMPs) also belong to the TGFβ family and are multifunctional growth factors involved in many aspects of tissue development and moφhogenesis including regulation of FSH action in the ovary

Using primary pituitary cell cultures derived from transgenic mice which carry the ovine FSHβ promoter linked to a luciferase reporter gene (oFSHβLuc), BMP-7 or BMP-6 was found to stimulate oFSHβLuc expression by 6-fold Also transient expression of the oFSHβLuc m a transformed gonadotrope cell line, LβT2, was induced 4-fold by BMP-7 or BMP-6 treatment Both BMP-7 and BMP-6 increased FSH secretion from LβT2 cells, demonstrating for the first time that a functional BMP system is present m gonadotropes Two neutralizing antibodies to BMP-7, which cross-react with BMP-6 but not with activm A, decreased the basal expression of oFSHβLuc in transgenic mouse pituitary cultures by 80- 90%, suggesting an autocrme or paracrme role for BMP-7 or BMP-6 in FSH synthesis

Neither bio-neutrahzmg antibody to activm A or activm B decreased basal oFSHβLuc expression significantly Furthermore, mRNAs for BMP-7 and BMP-6 were detected in mouse pituitaπes using RT-PCR These results indicate that BMP-7 and BMP-6 can function as FSH stimulators and may be significant physiological factors maintaining basal FSH expression

Example 4 Morphogen and GnRH Regulation of FSHβ

Expression of folhcle-stimulatmg hormone (FSH) depends on gonadotropin releasing hormone (GnRH), and part of this regulation is thought to occur directly and selectively at

FSHβ transcπption Although difficult, it has been shown that GnRH can induce FSHβ transcription by 2- to 3-fold in vivo and in tissue culture of primary gonadotropes or non- gonadotropes fortified with GnRH receptors

Studies with non-gonadotropes have identified two highly conserved AP-1 sites in the proximal promoter (-120 bp and -83 bp) of the ovine FSHβ gene as being important for induction bv GnRH To study the significance of these AP-1 sites in primary gonadotropes, transgenic mice were produced that express luciferase under control of 4 7 kb of the ovine FSHβ promoter (oFSHβLUC) with or without functioning AP-1 sites (-120/-83) Luciferase was expressed in these mice (+/- AP-1 sites) only in the pituitary and regulated in vivo as if it were FSHβ, itself (+/- AP-1 sites). Using static cultures of pituitaπes from these mice, it was possible to show a 2- to 3-fold stimulation of luciferase expression by GnRH in wild-type cultures, but not in cultures expressing the AP-1 mutant oFSHβLUC. These results link the AP-1 sites to GnRH induction of FSHβ transcription. However, since expression of oFSHβLUC (mutant or wild-type) reflected normal FSHβ expression in all transgenic mouse lines, it appears that direct transcriptional regulation of FSHβ by GnRH in vivo may be relegated to subtle changes that are likely to be important but not yet understood. Our studies on GnRH eventually led to a focus on activin and other Moφhogensthat had larger effects on FSHβ transcription than GnRH. These studies indicated that activin A, as well as

BMP6 and BMP7 could stimulate FSHβ transcription 8- to 12-fold. Future studies will define activin/BMP response element(s) and use transgenic technology to determine the physiological relevance of activin/BMPs to reproductive function.

In conclusion, these studies, taken together with those of others, suggest that GnRH regulates FSH expression primarily at a global level of gonadotrope "well being" rather than at the micro-management level of specifically altering FSHβ transcription. By contrast, Moφhogens more likely to influence FSHβ directly and selectively at the transcriptional level. Support came from NIH grant HD 34863 and gifts of BMP reagents from Dr. P.L. Kaplan & Dr. D.M. Bosukonda, Creative Biomolecules, Inc., Hopkinton, MA 01748

The foregoing description has been presented only for the puφoses of illustration and is not intended to limit the invention to the precise form disclosed, but by the claims appended hereto. In the specification and the appended claims, the singular forms include plural references, unless the context clearly dictates otherwise. All patents and publications cited in this specification are incoφorated by reference.

Claims

What is claimed is

1 A method for increasing fertility in a subject, comprising

administering to the subject a therapeuticaUy effective amount of a moφhogen, the moφhogen comprising an ammo acid having a sequence selected from the group consisting of a sequence

(a) having at least 70% homology with the C-termmal seven-cysteme skeleton of human OP-1, ammo acids 330-431 of SEQ ID NO 2,

(b) having greater than 60% ammo acid sequence identity with said C-termmal seven-cysteme skeleton of human OP- 1 ,

(c) defined by SEQ ID NO 5,

(d) defined by SEQ ID NO 6,

(e) defined by SEQ ID NO 7,

(f) defined by SEQ ID NO 8, and

(g) defined by OPX, SEQ ID NO 3

A method for alleviating symptoms of menopause, comprising

administering to the subject a therapeuticaUy effective amount of a moφhogen, the moφhogen comprising an amino acid having a sequence selected from the group consisting of a sequence

(a) having at least 70% homology with the C-termmal seven-cysteme skeleton of human OP-1, ammo acids 330-431 of SEQ ID NO 2, (b) having greater than 60% ammo acid sequence identity with said C-termmal seven-cysteine skeleton of human OP-1,

(c) defined by SEQ ID NO: 5,

(d) defined by SEQ ID NO- 6,

(e) defined by SEQ ID NO: 7,

(f) defined by SEQ ID NO: 8; and

(g) defined by OPX, SEQ ID NO: 3

A method for delaying the onset of menopause, compπsing-

administering to the subject a therapeuticaUy effective amount of a moφhogen, the moφhogen comprising an ammo acid having a sequence selected from the group consisting of a sequence:

(a) having at least 70% homology with the C-termmal seven-cysteine skeleton of human OP-1, ammo acids 330-431 of SEQ ID NO: 2;

(b) having greater than 60%> ammo acid sequence identity with said C-terminal seven-cysteine skeleton of human OP- 1 ;

(c) defined by SEQ ID NO: 5;

(d) defined by SEQ ID NO: 6,

(e) defined by SEQ ID NO" 7;

(f) defined by SEQ ID NO: 8; and

(g) defined by OPX, SEQ ID NO. 3

A method for contraception, comprising administering to the subject a therapeuticaUy effective amount of a compound that interferes with the binding of a moφhogen to its receptor, the moφhogen comprising an amino acid having a sequence selected from the group consisting of a sequence:

(a) having at least 70% homology with the C-terminal seven-cysteine skeleton of human OP-1, amino acids 330-431 of SEQ ID NO: 2;

(b) having greater than 60% amino acid sequence identity with said C-terminal seven-cysteine skeleton of human OP-1;

(c) defined by SEQ ID NO: 5;

(d) defined by SEQ ID NO: 6;

(e) defined by SEQ ID NO: 7;

(f) defined by SEQ ID NO: 8; and

(g) defined by OPX, SEQ ID NO: 3.

5. The method of claim 4, wherein the compound that interferes with the binding of the moφhogen and its receptor is an anti-moφhogen antibody.

6. The method of claim 4, wherein the compound that interferes with the binding of the moφhogen and its receptor is an anti-receptor antibody.

7. The method of claim 4, wherein the compound that interferes with the binding of the moφhogen and its receptor is a moφhogen receptor antagonist.

8. The method of claim 1-4, wherein the moφhogen induces estrogen synthesis by the ovary of the subject.

9. The method of claim 1-4, wherein the administration of the moφhogen attenuates progesterone synthesis by the ovary of the subject.

10. The method of claim 1 -4, wherein the subject has healthy ovary follicles. The method of claim 1-4, wherein the subject has atretic ovary follicles

The method of claim 1 -4, wherein the subject has both atretic and healthy follicles

The method of claim 1-4, wherein the administration of the moφhogen attenuates progesterone synthesis by the ovary of the subject

The method of claim 1 -4, wherein the administration of the moφhogen attenuates progesterone synthesis by the ovary of the subject

The method of claim 1-4, wherein the moφhogen is selected from the group consisting of OP-1, OP-2, OP-3, BMP-2, BMP-3, BMP-3b, BMP-4, BMP-5, BMP-6, BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-15, GDF-1, GDF-2, GDF-3, GDF-5, GDF-6, GDF-7, GDF-8, GDF-9, GDF-10, GDF-1 1, GDF-12, 60A, dpp, Vgl,

Vgr-1 , NODAL, UNIVIN, SCREW, ADMP, and NEURAL, and moφhogemcally-active ammo acid vaπants of any thereof

The method of claim 1-4, wherein the moφhogen is OP-1

The method of claim 1-4, wherein the administration of the moφhogen increases ovaπan synthesis of estrogen