JP2008521891A - Regulation of ovulation - Google Patents

Abstract

  The present invention identifies novel domains that are functionally important in GDF-9 and GDF-9B molecules, and interacts with the domains to modulate the biological activity of these molecules, thereby regulating mammalian ovarian function and The present invention relates to peptides and antibodies for changing the ovulation rate.

Description

  The present invention identifies novel functionally important domains on GDF-9 and GDF-9B molecules, and interacts with them to modulate the biological activity of these molecules, thereby regulating mammalian ovarian function and ovulation. It relates to agonists and antagonists for changing the rate.

  GDF-9 and GDF-9B (also known as BMP15) are expressed in the oocytes of developing follicles and are involved in mammalian fertility (Fitzpatrick et al., 1998). ). GDF-9 is a member of the transforming growth factor beta (TGFβ) superfamily (McPherron and Lee, 1993) and is expressed in oocytes from the early stages of follicular development to ovulation. (McGrath et al., 1995; Laitinen et al., 1998). GDF-9B is closely related to GDF-9 (Dube et al., 1998; Laitinen et al., 1998) and is expressed in mouse oocytes at the same time as GDF-9, but human It is expressed slightly later than GDF-9 in the primary follicle. GDF-9 and GDF-9B in the ovaries are now human (Aaltonen et al., 1999), rodents (Laitinen et al., 1998; Dube et al., 1998; Yatinen (Jatinen et al., 1999), ruminants (Bodensteiner et al., 1999; Bodensteiner et al., 2000; Galloway et al., 2000) and marsupial (Eckery) Et al., 2002) have been found to be expressed in developing oocytes. In sheep, GDF-9 expression is found in primordial follicles, whereas GDF-9B is expressed in primary follicles (Bodensteiner et al., 1999; Galloway et al., 2000).

  GDF-9 and GDF-9B, like most other members of the TGFβ family, are encoded as prepropeptides containing a signal peptide, a pro region and a C-terminal mature region that is a biologically active peptide. Cleavage of the mature region from the pro region is performed by intracellular furin-like proteases and occurs at conserved furin protease cleavage sites. Most members of the TGFβ superfamily show biological activity as dimers, and GDF-9 and GDF-9B do not contain cysteine molecules responsible for covalent interchain disulfide bonds found in almost all family members These molecules are believed to exhibit biological activity as dimers (Galloway et al., 2000; Yan et al., 2001). However, the dimer showing bioactivity is a homodimer (GDF9-GDF9 and GDF9B-GDF9B) or a heterodimer (GDF9-GDF9B), or all three dimer forms are all It's not clear if they will be involved. Based on the above model, it is hypothesized that GDF-9 homodimer plays a more important role in mice but GDF-9B homodimer has the highest biological activity in sheep (Yan et al., 2001). Year). It is unclear whether any of these differences are related to the fact that sheep are single ovulators (usually mature only one egg per cycle) while mice are polyovulatory animals. . Clearly, both GDF-9 and GDF-9B play an important role in the control and maintenance of fertility in mammals, and understanding the nature of their action is essential for therapeutic development.

  In Jeffery et al., 2003, a bioinformatics tool (GoCore) was used to identify GDF-9 between various TGF-β family members and between different species with the intention of identifying functionally important regions. The storage area in GDF-9B was analyzed. However, this study did not correlate with regions identified as sites that could be functionally important with an effect on the regulation of ovulation in vivo.

  In vivo by identifying functionally important domains for GDF-9 and / or GDF-9B molecules and interacting with such domains or mimetics of such domains and modulating their biological activity It is an object of the present invention to identify agonists and antagonists that modulate ovarian function and ovulation rate in mammals and / or to provide publicly useful options.

  As used herein and in the claims, the term “comprising” means “consisting at least in part of”, ie, an independent claim containing this term. In that case, all features prefaced by this term in each claim need to be present, meaning that other features may also be present.

SUMMARY OF THE INVENTION The present invention relates to agonists and antagonists based on the identification of functionally important novel domains on GDF-9 and GDF-9B molecules.

More particularly, the present invention is based on the identification and characterization of functionally important domains near the N-terminus of mature GDF-9 and GDF-9B molecules, wherein the N-terminal domain of GDF-9 is an amino acid sequence:
DQESASSELKKPLVPPASVNLSEYFKQFLFPQNEC (SEQ ID NO: 1); and the N-terminal domain of GDF-9B has the amino acid sequence:
QAGSIASEVPGPSREHDGPESNQC (SEQ ID NO: 2);

  Accordingly, the present invention relates to an isolated fragment of the N-terminal domain of GDF-9 capable of regulating female mammal ovulation, wherein the N-terminal domain of GDF-9 is the amino acid sequence DQASSASSLKPLVPPASVNLSEYFKQFLFPQNEC (SEQ ID NO: 1) or its It consists of a functional derivative, homologue, analogue or mimetic.

  Preferably, the peptide fragment comprises at least one amino acid from positions 1-9 or 25-34 of SEQ ID NO: 1.

  The present invention also relates to an isolated peptide fragment of the N-terminal domain of GDF-9B that can regulate ovulation in female mammals, wherein the N-terminal domain of GDF-9B is the amino acid sequence QAGSIASEVPGPSREHDGPESNQC (SEQ ID NO: 2) or its It consists of a functional derivative, homologue, analogue or mimetic.

  Preferably, the peptide fragment comprises at least one amino acid from positions 1-5 or 21-23 of SEQ ID NO: 2.

  The amino acid symbols correspond to the single letter codes shown in Table 1 below.

  The isolated peptide fragment of the invention preferably comprises at least 5 contiguous amino acids of SEQ ID NO: 1 or SEQ ID NO: 2 or functional derivatives, homologues, analogs or mimetics thereof. More preferably, the peptide fragment is at least 8, at least 10, at least 12, at least 14, at least 16, at least 18 or at least 20 contiguous amino acids of SEQ ID NO: 1 or SEQ ID NO: 2 or their functions A derivative, homologue, analogue or mimetic.

Most preferably, the peptide fragment of the present invention is
DQEASSSELKKPLV (C) (SEQ ID NO: 3),
SEYFKQFLFPQNEC (SEQ ID NO: 4),
QAGSIASEVPGPSR (C) (SEQ ID NO: 5), and SREHDGPESNQC (SEQ ID NO: 6),
Or selected from the group comprising functional derivatives, homologues, analogues or mimetics thereof.

  Amino acids in parentheses indicate amino acids that are optionally added for binding purposes.

  The invention also relates to antibodies or antibody fragments that bind to one or more peptide fragments of the invention.

  In a further aspect, the present invention provides a method of modulating the ovulation rate of a female mammal, said method interacting with the N-terminal domain of GDF-9 and / or GDF-9B as defined above, And administering to said mammal an effective amount of one or more isolated peptide fragments and / or antibodies or antibody fragments of the invention capable of altering their biological activity.

  Preferably, the present invention provides a female mammal with an effective amount of one or more peptides selected from SEQ ID NOs: 3-6 or functional variants thereof and / or antibodies or antibody fragments that bind to them. A method of regulating the ovulation rate of said mammal comprising the step of administering is provided.

  In a further aspect, the present invention provides the use of one or more isolated peptide fragments of the present invention and / or antibodies or antibody fragments that bind to them in the manufacture of a medicament for regulating the ovulation rate of a female mammal. I will provide a.

  Preferably, the present invention provides one or more peptides selected from the group comprising SEQ ID NOs: 3 to 6 or functional variants thereof and / or in the manufacture of a medicament for regulating the ovulation rate of female mammals. Use of antibodies or antibody fragments that bind to them is provided.

  In yet another aspect, the invention relates to one of the inventions capable of interacting with the N-terminal domain of GDF-9 and / or GDF-9B as defined above together with a pharmaceutically acceptable carrier or excipient. Alternatively, a pharmaceutical composition comprising a plurality of types of isolated peptide fragments is provided.

  Preferably, the composition comprises one or more peptides selected from the group comprising SEQ ID NOs: 3-6 or functional variants thereof together with a pharmaceutically acceptable carrier or excipient. The composition may additionally or alternatively include an antibody or antibody fragment that binds to one or more of the peptides together with a pharmaceutically acceptable carrier or excipient.

DESCRIPTION OF THE DRAWINGS The present invention will now be described in detail with reference to the drawings in the following accompanying drawings:
FIG. 1 shows the position of the peptides of SEQ ID NO: 3 and 4 on GDF-9;
FIG. 2 shows the position of the peptides of SEQ ID NO: 5 and 6 on GDF-9B;
Figures 3a and 3b show the sequence homology of GDF9 with a number of different species and the consensus sequence of the N-terminal domain;
FIG. 4 shows the effect of treatment with GDF-9 and GDF-9B peptides on bovine antral follicle development (A = control, mussel hemocyanin (KLH) only; B = KLH-GDF-9 (SEQ ID NO: 7)) C = KLH-GDF-9B (SEQ ID NO: 5); D = KLH-GDF-9 and KLH-GDF-9B (SEQ ID NO: 5 and 7);
FIG. 5 shows a polyclonal antibody derived from sheep immunized against sheep GDF-9 peptide (SEQ ID NO: 3 or SEQ ID NO: 4) or GDF-9B peptide (SEQ ID NO: 5) when added directly to a bioassay. Shows the effect of rat granulosa cells stimulated with ovine (o) GDF-9 and oGDF-9B on ³ H-thymidine incorporation. The horizontal line shows the response level of granulosa cells treated with control medium (ie medium without oGDF-9 or oGDF-9B). The data shown is the mean of 4 replicate experiments and the standard error of the mean. For treated with control medium cells ^{* P <0.05, ** P <} 0.01, do not receive antibody, for cells treated with oGDF-9 and oGDF-9B ^a P <0.05, ^bP <0.01;
FIG. 6 shows mouse (m) GDP-9 and sheep (o) GDF of a polyclonal antibody derived from sheep immunized against sheep GDF-9 peptide (SEQ ID NO: 4) when added directly to the bioassay. It shows the effects on ³ H- thymidine incorporation stimulated rat granulosa cells in-9b. The horizontal line shows the level of response of granulosa cells treated with control medium (ie medium without mGDF-9 or oGDF-9B). The data shown is the average of three replicate experiments and the standard error of the average. ^* P <0.05, ^** P <0.01 for cells treated with control medium, ^b P for cells treated with mGDF-9 and oGDF-9B without antibody <0.01.
FIG. 7 shows stimulation of sheep-derived polyclonal antibodies immunized against sheep GDF-9 peptide (SEQ ID NO: 4) with sheep (o) GDF-9 and oGDF-9B when added directly to the bioassay. ³ shows the effect of different doses on ³ H-thymidine uptake of cultured rat granulosa cells. The data shown is the mean of 4 replicate wells from one pool of granulosa cells and the standard error of the mean.
FIG. 8 shows stimulation of a sheep-derived polyclonal antibody immunized against sheep GDF-9B peptide (SEQ ID NO: 5) with sheep (o) GDF-9 and oGDF-9B when added directly to the bioassay. ³ shows the effect of different doses on ³ H-thymidine uptake of cultured rat granulosa cells. The data shown is the mean of 4 replicate wells from one pool of granulosa cells and the standard error of the mean.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides novel functionally important domains at the N-terminus of GDF-9 and GDF-9B. It is hypothesized that stimulation or inhibition of this domain by agonists or antagonists that interact with this domain is effective in regulating the ovulation rate of female mammals.

  For the sequence of the N-terminal domain, the corresponding sequences of GDF-9 and GDF-9B proteins in a number of different species were compared with the determined consensus sequences shown in FIGS. 3a and 3b for GDF-9 and GDF-9B, respectively. .

  A number of which are then expected to have agonistic or antagonistic effects on the biological activity of GDF-9 and / or GDF-9B when corresponding to sequences in the N-terminal domain and when administered in vivo. Peptides were synthesized. In particular, present outside the molecule according to the three-dimensional structure of the peptide; present in the mobile region of the molecule; has a length of at least 9 amino acids; is non-homologous to other TGFbeta family members; The peptides were designed to be non-convergent with the known proteins; in regions that do not contain glycosylation sites; and capable of binding to carrier proteins. Combining these elements was thought to produce peptides that are highly specific across species and have no cross-reactivity problems.

  Accordingly, in a first embodiment, the present invention relates to an isolated fragment of the N-terminal domain of GDF-9 that can regulate female mammal ovulation, wherein the N-terminal domain of GDF-9 is the amino acid sequence DQASSASSELKPLVPPASVNLSEYFKQFLFPQNEC (SEQ ID NO: 1). The amino acid symbols correspond to the single letter codes shown in Table 1 below.

  Preferably, the isolated peptide fragment comprises at least one amino acid at positions 1-9 or 25-34 of SEQ ID NO: 1.

  Preferably, the peptide comprises at least 5 contiguous amino acids of SEQ ID NO: 1 or functional derivatives, homologues, analogs or mimetics thereof. More preferably, the peptide comprises at least 8, at least 10, at least 12, at least 14, at least 16, at least 18 or at least 20 contiguous amino acids of SEQ ID NO: 1 or functional derivatives, homologues thereof. , Including analogs or mimetics.

Most preferably, the peptide is
DQESASSELKKPLV (C) (SEQ ID NO: 3), and SEYFKQFLFPQNEC (SEQ ID NO: 4)
Or selected from the group comprising functional derivatives, homologues, analogues or mimetics thereof. Amino acids in parentheses indicate amino acids that are optionally added for binding purposes.

  The present invention also relates to antibodies or antibody fragments that bind to one or more GDF-9 peptides of the present invention.

In a second embodiment, the present invention relates to an isolated peptide fragment of the N-terminal domain of GDF-9B capable of regulating female mammal ovulation, wherein the N-terminal domain of GDF-9B has the amino acid sequence:
QAGSIASEVPGPSREHDGPESNQC (SEQ ID NO: 2)
Or a functional derivative, homologue, analogue or mimetic thereof. The amino acid symbols correspond to the single letter codes shown in Table 1 below.

  Preferably, the isolated peptide fragment comprises at least one amino acid at positions 1-5 or 21-23 of SEQ ID NO: 2.

  Preferably, the peptide comprises at least 5 contiguous amino acids of SEQ ID NO: 2 or functional derivatives, analogs, homologues or mimetics thereof. More preferably, the peptide comprises at least 8, at least 10, at least 12, at least 14, at least 16, at least 18 or at least 20 contiguous amino acids of SEQ ID NO: 2 or functional derivatives thereof, homologues Including body, analog or mimetic.

Most preferably, the peptide is
QAGSIASEVPGPSR (C) (SEQ ID NO: 5), and SREHDGPESNQC (SEQ ID NO: 6),
Or selected from the group comprising functional derivatives, homologues, analogues or mimetics thereof.

  The present invention also provides an antibody or antibody fragment that binds to one or more GDF-9B peptides of the present invention.

  Amino acids in parentheses indicate amino acids that are optionally added for binding purposes.

  The peptides of the present invention can be synthesized using known techniques.

  Analogs, derivatives or variants of the peptides of the present invention may contain sequence modifications or non-sequence modifications. Non-sequence modifications can include acetylation, methylation, phosphomethylation, carboxylation or glycosylation.

  Specific N-terminal peptides exemplified in the present invention are shown in relation to their position on the N-terminal portion of GDF-9 and GDF-9B in FIGS. 1 and 2, respectively.

  Preferred analogs include peptides having sequences that differ from the sequences of the invention by one or more conservative amino acid substitutions, deletions or insertions that do not affect the biological activity of the peptide. Conservative substitutions are typically substitutions of one amino acid with another amino acid having characteristics similar to, for example, valine, glycine; glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine Serine, threonine; lysine, arginine; and substitution within the group of phenylalanine, tyrosine. Examples of conservative substitutions can also be found within the sequences of GDF-9 and GDF-9B in FIGS. 3a and 3b, whereby substitutions in different mammalian species are shown relative to the consensus sequence. Other conservative substitutions can be obtained from Table 1 below.

  Other analogs include peptides with modifications that affect the stability of the peptide. Such analogs can include, for example, one or more non-peptide bonds (which replace peptide bonds) within the peptide sequence. Also included are analogs including residues other than naturally occurring L-amino acids, such as D-amino acids, or non-naturally occurring synthetic amino acids, such as β or γ amino acids and cyclic analogs.

  In a further aspect, the present invention provides the use of the agonists and antagonists of the present invention in a method of modulating the ovulation rate of female mammals, including human and non-human mammals. Such non-human mammals include non-human primates such as sheep, cows, goats, deer, pigs, horses, camelids, possums, marmosets, cats, dogs and other commercially important species.

  The method can include administering to the mammal an effective amount of one or more agonists or antagonists, or functional variants thereof, or an antibody or antibody fragment that binds to them.

  It is believed that N-terminal domain peptides corresponding to the particular mammalian GDF-9 and GDF-9B to be treated will be used in the methods of the invention. Examples of GDF-9 and GDF-9B peptides from different species corresponding to the specific N-terminal peptides of SEQ ID NOs: 3-6 are shown in Table 1a below.

  Amino acid alignments of multiple species of mature GDF-9 and GDF-9B are shown in FIGS. 3a and 3b. These alignment sequences were generated using multiple alignment programs of Vector NTI. This program uses the ClustalW algorithm (Thompson et al., 1994). A region encoding the mature region of the protein was identified and used to generate a protein sequence in which the nucleotide sequence was predicted. The sequences used for alignment (GenBank accession numbers) are as follows: GDF-9: cattle (NM_174648), goat (AH014112), sheep (AF0785545), pig (NM_001001909), dog (XM_538624), cat (in-house database), fox mouse (AY033826), rabbit (in-house database), mouse (NM_008110) , Rat (NM_021672), chimpanzee (XM_527008) and human (NM_005260). GDF-9B: cattle (AY5722412), deer (in-house database), dog (XM_549005), cat (in-house database), black rat (AH012378), chimpanzee (XM_529247), human (AF0835030), mouse (NM_021670), rabbit (in-house database) ), Pig (AF458070), goat (in-house database) and sheep (AF236079).

  The regulation of ovulation rate is such that administration of an agonist or antagonist of the present invention to a female mammal results in the production of antibodies against the agonist or antagonist in vivo, followed by GDF-9 and / or GDF-9B. May include increasing or decreasing the ovulation rate of the animal by binding to its N-terminal domain and affecting their biological activity.

  Without being bound by theory, GDF-9 by an agonist / antagonist and in particular one or more antibodies raised against one or more peptides selected from the group comprising SEQ ID NO: 3-6 It is believed that binding of the N-terminal domain in and / or GDF-9B results in changes in circulating concentrations of GDF-9 and / or GDF-9B that exhibit biological activity. If such a decrease includes an approximately 50% decrease in GDF-9B activity, an increase in ovulation rate has been observed so far, particularly in the case of Hannah sheep, the result of a single point mutation in the GDF-9B gene. The amount of active GDF-9B in heterozygous animals should have been halved and the ovulation rate and twin birth should have increased. In homozygous animals, the animals were infertile when there was no or very little circulation of active GDF-9B (Galloway et al., 2000). Similarly, point mutations in the GDF-9 gene and concomitant regulation of ovulation rates have been observed in sheep (WO 03/102199; Hanrahan et al., 2003).

  Thus, an agonist or antagonist of the present invention that results in an approximately 50% decrease in circulating concentrations of active GDF-9 and / or GDF-9B results in an increase in ovulation rate, while active GDF-9 and It has been hypothesized that agonists or antagonists that produce a reduction to about zero in circulating concentrations of GDF-9B will result in reduced ovulation and sterility in female mammals.

Preferably, the agonist or antagonist of the present invention is an antibody that binds to the consensus N-terminal domain of GDF-9 and / or GDF-9B of SEQ ID NOs: 1 and 2. The term “antibody” includes a fragment or similar of an antibody that retains the ability to bind to a consensus binding domain as defined herein, including but not limited to Fv, Fc, F (ab) ₂ fragments, ScFv molecules, and the like. It should be understood to encompass the body. The antibody can be polyclonal or monoclonal, but is preferably monoclonal. Such antibodies can be prepared by any technique known in the art (eg, Jüngel et al., 2002) intended for administration to animals, ie, passive immunization. Alternatively, such antibodies can be produced in vivo by administration of an antigen in a suitable adjuvant, ie, intended for use in active immunization. Suitable adjuvants include Freund's complete or incomplete adjuvant or similar immunostimulants. The antibodies of the invention can be produced by genetic engineering methods such as chimeric and humanized monoclonal antibodies, including both human and non-human portions, that can be produced using standard recombinant DNA techniques. It is.

  The present invention further relates to the combined use of one or more agonists and / or antagonists of the present invention and one or more active ingredients known to increase the effect on ovulation by regulating the ovulation rate. Be considered. The active ingredient may be selected from the group comprising GDF-9, GDF-9B, BMPRII, BMPIB receptor (ALK6), ALK5 and BMP6, or functional fragments or variants thereof. In particular, the present invention examines the combined use of an antibody or antibody fragment (Fc) that binds to the peptide of SEQ ID NO: 5 or 6 with the BMP1B receptor in the regulation of ovulation in female mammals.

  The present invention further provides a pharmaceutical composition comprising at least one agonist or antagonist of the present invention together with a pharmaceutically acceptable carrier useful for modulation of ovulation rate.

  It is contemplated that the biological activity in the agonists or antagonists of the present invention is tested in animal models or in vitro models, and compounds that exhibit the appropriate activity are formulated in pharmaceutical compositions. In addition to one or more agonists or antagonists of the present invention, the pharmaceutical composition of the present invention may be a pharmaceutically acceptable excipient, carrier, buffer, stabilizer, or other well known in the art. It can contain substances. Such materials should be non-toxic and do not interfere with the effectiveness of the active ingredient. The exact nature of the carrier or other material will depend on the desired properties of the pharmaceutical composition and the route of administration, eg oral, intravenous, dermal, subcutaneous, intradermal, intramuscular or intraperitoneal.

  Pharmaceutical compositions for oral administration can be in tablet, lozenge, capsule, powder, powder or liquid form. Tablets or other solid oral dosage forms will typically include a solid carrier such as gelatin, starch, mannitol, crystalline cellulose, or other inert materials commonly used in pharmaceutical manufacture. Similarly, liquid pharmaceutical compositions such as syrups or emulsions will generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil.

  For intravenous, cutaneous, subcutaneous, intradermal or intraperitoneal injection, the active ingredient is pyrogen free and exists in the form of a parenterally acceptable aqueous solution with suitable pH, isotonicity and stability It will be.

  In a further embodiment, the present invention provides one or more additional ovulators of ovulation, wherein the co-administration with the pharmaceutical composition of the present invention provides an additive or synergistic effect on the treatment regime. ). Examples of such additional regulators of ovulation include follicle stimulating hormone, Androidvax (androstenedione protein conjugate vaccine), and steroid hormones. Such modulators, as will be understood by those skilled in the art, are divided, continuous or simultaneous with the at least one agonist or antagonist of the present invention, depending on whether ovulation is to be promoted or suppressed. Can be administered.

  Administration of the pharmaceutical composition of the present invention is preferably performed in a “therapeutically effective amount” sufficient to exhibit the desired benefit to the individual. The actual dosage, as well as the frequency and time course of administration, will depend on the nature and severity of the basic symptoms in the female mammal. For example, treatment regimens such as dose-related decisions are within the responsibility of general physicians and other physicians, and are typically known to the disease requiring treatment, individual patient symptoms, delivery site, method of administration, and physicians. Other factors are considered. Examples of the techniques and protocols described above are described in Remington's Pharmaceutical Sciences, 16th edition, Oslo. (Oslo A.) (eds.), Can be found in 1980.

  The present invention will now be described in greater detail by reference to specific examples and should not be construed in any way, such as limiting the scope of the invention.

Example 1
Use of peptides and antibodies to the N-terminal domain of GDF-9 and GDF-9B to engineer ovulation of sheep Corresponding to the N-terminal domain of GDF-9 and GDF-9B protein sequences and, if necessary, mussel hemocyanin Four 12-15mer peptides were synthesized containing additional residues to promote binding to (Keyhole Lime Haemocyanin) (KLH) to generate an antigen. The peptide sequence is
DQEASSSELKKPLV (C) (SEQ ID NO: 3),
SEYFKQFLFPQNEC (SEQ ID NO: 4),
QAGSIASEVPGPSR (C) (SEQ ID NO: 5), and SREHDGPESNQC (SEQ ID NO: 6),
Met.

  In this study, a group of 10 estrus Romney ewes were injected with 0.4 mg / ewe of each peptide-KLH antigen conjugate in Freund's complete adjuvant, and 10 mice as control groups. Estrous romney ewes were injected with 0.4 mg / sheep KLH antigen. The animals were then boosted (subcutaneously) four times at monthly intervals with additional antigen (0.2 mg / sheep each time) in a Span / Tween / oil mixture, and vasectomy was performed. Oestrous activity was monitored over the breeding period using the made rams. The ovulation rate was assessed by laparoscope again over 4 consecutive estrous cycles and at the completion of the experiment after about 20-30 days. The average ovulation rate was determined by averaging these 5 observations for each ewes.

  All control ewes showed periodic estrous activity. The results of the effect of the peptides of SEQ ID NOs: 3-6 on the ovulation rate showed a decrease in the ovulation rate of the ewes treated with peptide compared to the control ewes, as shown in Table 2 below. The results correlate with a significant increase in the levels of anti-GDF-9 serum antibody and anti-GDF-9B serum antibody. After dilution of sheep plasma to 1: 20,000 as previously shown (Jugel et al., 2002), antibody levels were measured using an ELISA method.

  The peptides of SEQ ID NOs: 3-5 resulted in a decrease in the ovulation rate of most animals within 2 weeks after the first booster injection. The decrease in ovulation rate was maintained until the end of the experiment. The peptide of SEQ ID NO: 6 did not decrease so much until the third observation (after 3 booster injections), and only 50% of the animals decreased. However, by the last booster injection, 9 out of 10 animals had reduced ovulation rates (Table 3). Reduction of ovulation was assessed by observation of the ovary that the ovary has no externally recognizable corpus luteum. Ewes that were considered estrus as assessed by observation of the uterus were excluded from the results in Table 3.

  These data clearly show that a significant reduction in ovulation rate can be induced by administration of a peptide antigen specific for the N-terminal domain of GDF-9 or GDF-9B. In this particular example, this effect was caused by active immunity (ie, administration of an antigen that produces antibodies in vivo). However, it is expected that a similar effect will be seen by passive immunity, ie administration of the antibody itself.

Example 2
Use of peptides and antibodies against the N-terminal domain of GDF-9 and GDF-9B to manipulate bovine ovulation

Two 15mer peptides were synthesized that corresponded to the N-terminal domain of the bovine GDF-9 and GDF-9B protein sequences and optionally included additional residues to promote binding to KLH and generate antigen. . The peptide sequence is
DQESVSSELKKPLV (C) (SEQ ID NO: 7), and QAGSIASEVPGPSR (C) (SEQ ID NO: 5)
Met.

  Note that SEQ ID NO: 7 is a functional variant of SEQ ID NO: 3 containing a single amino acid change (amino acid at position 5 is A → V).

  In this study, a group of 10 Free-Jean cows (Friesian-cross heifers) was administered four 0.5 ml injections to each 0.4 mg / bovine peptide-KLH in 2 ml Freund's complete adjuvant. Antigen conjugates were injected; 10 were injected with 0.4 mg / bovine both peptide-KLH antigen conjugates, and 10 were injected with KLH antigen as a control group. The animals were then boosted twice subcutaneously with additional antigen (0.2 mg / cow each time) in a Span / Tween / Marcol mixture twice at monthly intervals. Approximately 2 weeks after the second boost, the animals were sacrificed and their ovaries were collected. Ovarian activity was assessed by visual inspection of the ovaries. Ovulation rate was determined based on the number of luteal bodies visible on the surface of the ovary. If there is more functional corpus luteum but it appears that the corpus luteum is not functioning by morphological criteria (small, lack of angiogenesis, pale white) or morphological criteria (small, lack of angiogenesis) , Corpus luteum) and endocrinological criteria (low levels of progesterone in blood samples at the time of ovary recovery and 2 weeks prior to recovery) determined that the corpus luteum had disappeared (thus ovulation rate) Score is not calculated). Follicular development was assessed on 5 μm tissue sections stained with hematoxylin and eosin.

  Peripheral venous blood samples were collected (10 ml with heparinized vacutainer) at the first time point and at the first and second boosts and again 12 days after the first boost and just before sacrifice. The plasma samples were analyzed for antibody titer and progesterone concentration.

  All cows appear to have normal ovarian activity prior to immunization. When treated with the peptide of SEQ ID NO: 5 alone or with the peptide of SEQ ID NO: 7, significant changes in ovarian activity were seen as judged by ovulation rate. Treatment with SEQ ID NO: 7 peptide alone showed 30% of two ovulations, which was not statistically significant to the number of animals examined, but at an unusually high rate. Interestingly, treatment with only the peptide of SEQ ID NO: 5 showed that the ovaries were deactivated in 3 animals, ie the ovulation rate was significantly reduced, similar to the results seen in sheep (Table 4 below). See).

  GDF-9B peptide bound to KLH (SEQ ID NO: 5), GDF-9 peptide bound to KLH (SEQ ID NO: 7), or GDF-9 peptide bound to KLH (SEQ ID NO: 7) and KLH at the time of recovery It was shown that ovaries from cows immunized in combination with GDF-9B peptide (SEQ ID NO: 5) bound to morphologically differed from ovaries from control cows immunized with KLH. It was. The ovaries of cows immunized with GDF-9 and / or GDF-9B had few recognizable follicular follicles on the surface. Similar observations were made in comparison with ovarian tissue sections (see FIG. 4). In this observation, the percentage of the total area occupied by the follicular space, the number of antral follicles per unit area and the follicles by quantifying on 5 μm tissue sections stained with hemotoxylin and eosin. The average size (area) was measured (see Table 5 below). Treatment with peptides SEQ ID NO: 5, SEQ ID NO: 7 or both reduced the total area of the sections occupied by the follicular cavity, the number of antral follicles per unit area and the average size of the follicle (P <0.01). (See Table 5 below). Therefore, it was clarified from these results that the ovulation rate can be lowered when immunization with the peptide fragment of the present invention is continued for a long time. In fact, some animals treated with the peptide of SEQ ID NO: 5 (GDF-9B) have been shown to have an increased incidence of ovarian deactivation compared to controls (see Table 4 above). .

  These results demonstrate that the N-terminal peptide is active in regulating bovine ovulation rate and follicular development. The dose administered was the same as in sheep, but in all cattle ovulation was increased rather than decreased. This may be due to a “sheep dose” resulting in insufficient immunization resulting in low circulating levels of about 50% of active GDF-9 and / or GDF-9B, and as a result, as described above Ovulation rate increases.

  This was confirmed by antibody titer data using a previously shown ELISA (Jüngel et al., 2002) with minor modifications. See Table 6 below. The antigen coated on the microtiter plate was 200 ng sheep GDF-9 mature protein or 100 ng sheep GDF-9B mature protein. The antibody was detected using a 1: 20,000 dilution of rabbit anti-bovine IgG. Sera from cows was diluted 1: 500. This is a 40-fold higher dilution than that required for sheep serum in Experiment 1 above, which indicates that the dose of GDF-9 and / or GDF-9B peptide administered to cattle is “insufficient” It was suggested that the dose was

  However, as described above, by continuing the immunization regime for a long period of time, there is a possibility that a decrease in the predetermined follicular development of the cow and a decrease in the ovulation rate may be induced.

  Higher doses than in cattle, ie doses sufficient to reduce circulating levels of active GDF-9 and / or GDF-9B to about 0, or prolonged immunization, will significantly increase bovine ovulation rates It is expected that it will decrease.

  These data clearly show that important regulation of ovulation rate and follicular follicle development can be induced by administration of peptide antigens specific for the N-terminal domain of GDF-9 and / or GDF-9B. This suggests that the N-terminal mature region of GDF-9 and GDF-9B is important for bovine biological activity. In this particular example, this effect was caused by active immunity (ie, administration of an antigen that produces antibodies in vivo). However, it is expected that a similar effect will be seen by passive immunity, ie administration of the antibody itself.

Example 3
Effects of antibodies raised against GDF-9 and GDF-9B peptide fragments on 3 ^H -thymidine uptake by rat granulosa cells in vitro The methods by which various antibody formulations have been shown previously (McNatty et al. , 2005) were tested for their neutralizing ability on the effects of sheep or murine GDF-9 and sheep GDF-9B on rat granulosa cells. A total of 100 μg / ml IgG consisting of IgG purified from sheep immunized with GDF-9 or GDF-9B peptide fragments under the balance of IgG purified from KLH-immunized sheep was added in each treatment. The antibodies were able to neutralize the effects of growth factors specific for them (see FIGS. 5 and 6). Furthermore, this response was shown to be dose dependent in the two antibody samples tested against sheep GDF-9 and GDF-9B (see P <0.001, see FIGS. 7 and 8).

  Proteins for thymidine uptake by rat granulosa cells (eg, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 4) with the effect of active immunization with these peptides in sheep (see Example 1) and cattle (see Example 2) The neutralization of the effect of sheep and mouse GDF-9 and sheep GDF-9B by antibodies raised against the peptide fragment of the N-terminal region of No. 5) is that this region of the protein is It is important for activity. These results also suggest that antibodies to the N-terminal domain of the mature region of GDF-9 and GDF-9B are effective in passively neutralizing GDF-9 and GDF-9B.

  While the invention has been described in some detail for purposes of clarity and understanding, various modifications and changes to the embodiments and methods described herein will be defined as defined in the appended claims. It will be apparent to those skilled in the art that this can be done without departing from the scope of the invention.

  The present invention provides compositions and methods for modulating ovulation rate and thereby fertility in female mammals, including humans.

References

1. McPherron AC, Lee SJ: GDF-3 and GDF-9: two new members of the transforming growth factor-beta superfamily containing a novel pattern of cysteines.J Biol Chem 268: 3444-3449, 1993.

2. McGrath SA, Esquela AF, Lee SJ: Oocyte-specific expression of growth differentiation factor-9. Mol Endocrinol 9: 131-136, 1995.

3. Laitinen M, Vuojolainen K, Jaatinen R, Ketola I, Aaltonen J, Lehtonen E, Heikinheimo M, Ritvos O: A novel growth differentiation factor-9 (GDF-9) related factor is co-expressed with GDF-9 in mouse oocytes during folliculogenesis. Mech Dev 78: 135-140, 1998.

4. Dube JL, Wang P, Elvin J, Lyons KM, Celeste AJ, Matzuk MM: The bone morphogenetic protein 15 gene is X-linked and expressed in oocytes. Mol Endocrinol 12: 1809-1817, 1998.

5. Jaatinen, R., Laitinen, MP, Vuojolainen, K., Aaltonen, J., Louhio, H., Heikinheimo, K., Lehtonen, E. and Ritvos, O: Localization of growth differentiation factor-9 (Gdf- 9) mRNA and protein in rat ovaries and cDNA cloning of rat GDF-9 and its novel homolog GDF-9B. Mol Cell Endocrinol 156: 189-193, 1999.

6. Aaltonen J, Laitinen M, Vuojolainen K, Jaatinen R, Horelli-Kuitunen N, Seppa L, Louhio H, Tuuri T, Sjoberg J, Butzow R, Hovatta O, Dale L, Ritvos O: Human growth differentiation factor- 9 ( GDF-9) and its novel homolog GDF-9B are expressed during early folliculogenesis. J Clin Endocrinol Metab 84: 2744-2750, 1999.

7. Bodensteiner, KJ, Clay, CM, Moeller, CL and Sawyer, HR: Molecular cloning of the ovine growth / differentiation factor-9 gene and expression of growth / differentiation factor-9 in ovine and bovine ovaries. Biology of Reproduction 60, 381-386, 1999.

8. Bodensteiner, KJ, McNatty, KP, Clay, CM, Moeller, CL and Sawyer, HR: Expression of growth and differentiation factor-9 in the ovaries of fetal sheep homozygous or heterozygous for the Inverdale prolificacy gene (FecX ^I ). of Reproduction 62: 1479-1485, 2000.

9. Galloway SM, McNatty KP, Cambridge LM, Laitinen MPE, Juengel JL, Jokiranta TS, McLaren RJ, Luiro K, Dodds KG, Montgomery GW, Beattie AE, Davis GH, Ritvos O: Mutations in an oocyte-derived growth factor gene (BMP15) cause increased ovulation rate and infertility in a dosage-sensitive manner.Nature Genetics 25: 279-283, 2000.

10. Eckery, DC, Whale, LJ, Lawrence, SB, Wilde, KA, McNatty, KP & Juengel, JL: Expression of mRNA encoding growth differentiation factor 9 and bone morphogenetic protein 15 during follicular formation and growth in a marsupial, the brushtail possum (Trichasurus vulpecula). Molecular & Cellular Endocrinology; 2002.

11. Yan, C., Wang, P., DeMayo, J., DeMayo, FJ, Elvin, J., Carino, C., Prasad, SV, Skinner, SS, Dunbar, BS, Dube, JL, Celeste, AJ and Matzuk, MM: Synergistic roles of bone morphogenetic protein 15 and growth differentiation factor 9 in ovarian function.Molecular Endocrinology 15: 854-866, 2001.

12. Fitzpatrick, SL, Sindon, DM, Shughue, PJ, Lane MV, Merchenthaler, IJ, and Frail, DF: Expression of growth differntiating factor-9 messenger ribnucleic acid in ovarian and non-ovarian rodent and human tissue.Endocrinology, 139 (5), 2577-2578, 1998.

13. Hanrahan, JP, Gregan, SM, Mulsant, P., Mullen, M., Davis, GM, Powell, R., Galloway, SM ,: Mutations in the genes for oocyte-derived growth factor GDF-9 and BMP- 15 are associated with both increased ovulation rate and sterility in Cambridge and Belcare sheep (Ovis aeries). Biol. Reprod, 2003.

14. Juengel, JL, Hudson, NL, Heath, DL, Smith, P., Reader, KL, Lawrence, SB, O'Connell, AR, Laitinen, MP, Cranfield, M., Groome, NP, Ritvos, O. , McNatty, KP ,: Growth differentiation factor 9 and bone morphogenic protein 15 are essential for ovarian follicular development in sheep. Biol. Reprod. 67 (b), 1777-1789, 2002.

15. Jeffery, L., Mottershead, DG, Myllymaa, S., Gilchrist, R., Groome, N., and Ritvos, O.,: Grouping of conserved regions (GoCore): Better prediction of function from sequences.Poster ESHRE Campus April, 2003.

16. Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice.Nucleic Acids Res. 1994 22: 4673-4680.

17. McNatty, KP, Juengel, JL, Reader, KL, Lun, S., Myllymaa, S., Lawrence, SB, Western, A., Meerasahib, MF, Mottershead, DG, Groome, NP, Ritvos, O., Laitinen, MPE (2005). Bone morphogenetic protein 15 and growth differentiation factor 9 co-operate to regulate granulosa cell function.Reproduction 129: 473-480.

All references are incorporated herein by reference in their entirety.

The positions of the peptides of SEQ ID NO: 3 and 4 on GDF-9 are indicated. The positions of the peptides of SEQ ID NOs: 5 and 6 on GDF-9B are indicated. Figure 3 shows the sequence homology of GDF9 with a number of different species and the consensus sequence of the N-terminal domain. Figure 2 shows the sequence homology of GDF-9B with a number of different species and the consensus sequence of the N-terminal domain. Shows the effect of treatment with GDF-9 and GDF-9B peptide on bovine follicular follicular development (A = control, mussel hemocyanin (KLH) only; B = KLH-GDF-9 (SEQ ID NO: 7); C = KLH -GDF-9B (SEQ ID NO: 5); D = KLH-GDF-9 and KLH-GDF-9B (SEQ ID NO: 5 and 7). Sheep when ovine-derived polyclonal antibodies immunized against sheep GDF-9 peptide (SEQ ID NO: 3 or SEQ ID NO: 4) or GDF-9B peptide (SEQ ID NO: 5) when added directly to a bioassay (o ) Effect of rat granulosa cells stimulated with GDF-9 and oGDF-9B on ³ H-thymidine uptake. The horizontal line shows the response level of granulosa cells treated with control medium (ie medium without oGDF-9 or oGDF-9B). The data shown is the mean of 4 replicate experiments and the standard error of the mean. For treated with control medium cells ^{* P <0.05, ** P <} 0.01, do not receive antibody, for cells treated with oGDF-9 and oGDF-9B ^a P <0.05, ^bP <0.01. Stimulation of a polyclonal antibody derived from sheep immunized against sheep GDF-9 peptide (SEQ ID NO: 4) with mouse (m) GDP-9 and sheep (o) GDF-9B when added directly to the bioassay It shows the effects on ³ H- thymidine incorporation by rat granulosa cells. The horizontal line shows the level of response of granulosa cells treated with control medium (ie medium without mGDF-9 or oGDF-9B). The data shown is the average of three replicate experiments and the standard error of the average. ^* P <0.05, ^** P <0.01 for cells treated with control medium, ^b P for cells treated with mGDF-9 and oGDF-9B without antibody <0.01. Rat granules stimulated with sheep (o) GDF-9 and oGDF-9B when added directly to a bioassay of a polyclonal antibody from sheep immunized against sheep GDF-9 peptide (SEQ ID NO: 4) The effect of different doses on membrane cell ³ H-thymidine uptake is shown. The data shown is the mean of 4 replicate wells from one pool of granulosa cells and the standard error of the mean. Rat granules stimulated with sheep (o) GDF-9 and oGDF-9B, when a polyclonal antibody derived from sheep immunized against sheep GDF-9B peptide (SEQ ID NO: 5) is added directly to the bioassay The effect of different doses on membrane cell ³ H-thymidine uptake is shown. The data shown is the mean of 4 replicate wells from one pool of granulosa cells and the standard error of the mean.

Claims (34)

  An isolated fragment of the N-terminal domain of GDF-9 capable of regulating ovulation in female mammals, wherein the N-terminal domain of GDF-9 is the amino acid sequence DQESASSELKKPLVPPASVNLSEYFKQFLFPQNEC (SEQ ID NO: 1) or a functional derivative thereof An isolated fragment consisting of a homologue, analogue or mimetic.   The isolated peptide fragment of claim 1, comprising at least one amino acid at positions 1-9 or 25-34 of SEQ ID NO: 1.   An isolated peptide fragment of the N-terminal domain of GDF-9B that can regulate ovulation in female mammals, wherein the N-terminal domain of GDF-9B is amino acid sequence QAGSIASEVPGPSREHDGPESNQC (SEQ ID NO: 2) or a functional thereof An isolated peptide fragment consisting of a derivative, homologue, analogue or mimetic.   The isolated peptide fragment of claim 3, comprising at least one amino acid at positions 1-5 or 21-23 of SEQ ID NO: 2.   An isolated peptide fragment of the N-terminal domain of GDF-9 capable of regulating female mammal ovulation, said fragment comprising at least one amino acid at positions 1-9 or 25-34 of SEQ ID NO: 1 Or an isolated peptide fragment comprising a functional derivative, homologue, analogue or mimetic thereof.   An isolated peptide fragment of the N-terminal domain of GDF-9B capable of regulating female mammal ovulation, said fragment comprising at least one amino acid at positions 1-5 or 21-23 of SEQ ID NO: 2. Or an isolated peptide fragment comprising a functional derivative, homologue, analogue or mimetic thereof.   The isolated peptide fragment according to any one of claims 1 to 6, comprising at least 5 contiguous amino acids of SEQ ID NO: 1 or SEQ ID NO: 2 or functional derivatives, homologues, analogues or mimetics thereof.   8. The isolated peptide fragment of claim 7, comprising at least 8 contiguous amino acids of SEQ ID NO: 1 or SEQ ID NO: 2 or functional derivatives, homologues, analogs or mimetics thereof.   8. The isolated peptide fragment of claim 7, comprising at least 10 contiguous amino acids of SEQ ID NO: 1 or SEQ ID NO: 2 or functional derivatives, homologues, analogs or mimetics thereof.   8. The isolated peptide fragment of claim 7, comprising at least 12 contiguous amino acids of SEQ ID NO: 1 or SEQ ID NO: 2 or functional derivatives, homologues, analogs or mimetics thereof.   8. The isolated peptide fragment of claim 7, comprising at least 14 contiguous amino acids of SEQ ID NO: 1 or SEQ ID NO: 2 or functional derivatives, homologues, analogs or mimetics thereof.   8. The isolated peptide fragment of claim 7, comprising at least 16 contiguous amino acids of SEQ ID NO: 1 or SEQ ID NO: 2 or functional derivatives, homologues, analogs or mimetics thereof.   8. The isolated peptide fragment of claim 7, comprising at least 18 contiguous amino acids of SEQ ID NO: 1 or SEQ ID NO: 2 or functional derivatives, homologues, analogs or mimetics thereof.   8. The isolated peptide fragment of claim 7, comprising at least 20 contiguous amino acids of SEQ ID NO: 1 or SEQ ID NO: 2 or functional derivatives, homologues, analogs or mimetics thereof. DQEASSSELKKPLV (C) (SEQ ID NO: 3),
SEYFKQFLFPQNEC (SEQ ID NO: 4),
QAGSIASEVPGPSR (C) (SEQ ID NO: 5), and SREHDGPESNQC (SEQ ID NO: 6),
15. The isolated peptide fragment or functional derivative, homologue, analogue or mimetic thereof according to any one of claims 1-14 selected from the group comprising   An antibody or antibody fragment that binds to one or more peptide fragments according to any one of claims 1-15.   A method for regulating the ovulation rate of a female mammal comprising the step of administering to said mammal an effective amount of one or more isolated peptide fragments according to any one of claims 1-15. ,Method.   17. A method of regulating the ovulation rate of a female mammal, comprising administering to said mammal an effective amount of one or more antibodies or antibody fragments of claim 16.   A method for regulating the ovulation rate of a female mammal, comprising one or more antibodies or antibody fragments according to claim 16 together with one or more antibodies according to any one of claims 1 to 15. Administering an effective amount of an isolated peptide fragment to said mammal.   A method for regulating the ovulation rate of a female mammal, comprising one or more peptide fragments selected from SEQ ID NOs: 3 to 6 or functional derivatives, homologues, analogues or mimetics thereof and / or them Administering an effective amount of an antibody or antibody fragment that binds to said mammal.   Use of one or more isolated peptide fragments according to any one of claims 1 to 15 in the manufacture of a medicament for regulating the ovulation rate of a female mammal.   Use of one or more antibodies or antibody fragments according to claim 16 in the manufacture of a medicament for regulating the ovulation rate of a female mammal.   In the manufacture of a medicament for regulating the ovulation rate of a female mammal, a) one or more isolated peptide fragments according to any one of claims 1 to 15 and b) according to claim 16. Use of one or more antibodies or antibody fragments, wherein the medicament is formulated with the intention of splitting, sequential or simultaneous administration of a) and b).   24. Use according to any one of claims 21 to 23, wherein the isolated peptide fragment is selected from the group comprising SEQ ID NO: 3-6 or functional derivatives, homologues, analogues or mimetics thereof.   A pharmaceutical composition comprising one or more isolated peptide fragments according to any one of claims 1 to 15 together with a pharmaceutically acceptable carrier or excipient.   A pharmaceutical composition comprising one or more antibodies or antibody fragments according to claim 16 together with a pharmaceutically acceptable carrier or excipient.   17. One or more isolated peptide fragments according to any one of claims 1 to 15 together with a pharmaceutically acceptable carrier or excipient and one or more antibodies according to claim 16 or A pharmaceutical composition comprising an antibody fragment.   One or more peptides selected from the group comprising SEQ ID NOs: 3 to 6 or functional variants thereof together with pharmaceutically acceptable carriers or excipients and / or antibodies or antibody fragments that bind to them A pharmaceutical composition comprising.   A method for regulating the ovulation rate of a female mammal, wherein an effective amount of the composition according to any one of claims 25 to 28 is applied to GDF-9, GDF-9B, BMPRII, BMPIB receptor (ALK6). , Administering to said mammal in combination with one or more compounds selected from the group comprising ALK5 and BMP6, or functional fragments or variants thereof.   A method for regulating the ovulation rate of a female mammal, wherein an effective amount of an antibody or antibody fragment (Fc) that binds to any one peptide of SEQ ID NOs: 3 to 6 is used in combination with a BMP1B receptor (ALK6). Administering to said mammal.   29. A method for regulating the ovulation rate of a female mammal, wherein the effective amount of the composition according to any one of claims 25 to 28 is selected from the group consisting of FSH, androvax and steroid hormones. Administering to said mammal in combination with.   In the manufacture of a medicament for regulating the ovulation rate of a female mammal, a) a composition according to any one of claims 25 to 28 and b) a GDF-9, GDF-9B, BMPRII, BMPIB receptor ( Use of one or more compounds selected from the group comprising ALK6), ALK5 and BMP6 or functional fragments or variants thereof, wherein said agent is a split, sequential or simultaneous administration of a) and b) It is formulated with the intention of use.   In the manufacture of a medicament for regulating the ovulation rate of a female mammal, a) an antibody or antibody fragment (Fc) that binds to any one peptide of SEQ ID NOs: 3 to 6 and b) a BMP1B receptor (ALK6) Use, wherein the agents are formulated with the intention of dividing, sequential or simultaneous administration of a) and b).   In the manufacture of a medicament for regulating the ovulation rate of a female mammal, a) an ovulation selected from the group consisting of a composition according to any one of claims 25 to 28 and b) FSH, androvax and a steroid hormone. Use of a modulating agent, wherein said agent is formulated with the intention of splitting, continuous or simultaneous administration of a) and b).

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