60/530, 220, filed December 16,2003, which application is incorporated herein by reference in its entirety GOVERNMENT RIGHTS This invention was made with government support under federal grant no.

HD20788 awarded by the National Institutes of Health. The United States Government may have certain rights in this invention.

INTRODUCTION Background of the Invention At the beginning of the reproductive cycle in mammals, follicle stimulating hormone (FSH) and luteinizing hormone (LH) are secreted from the pituitary gland and stimulate growth of the ovarian follicles containing the ova. As the follicles grow and develop, estrogen hormones (principally estradiol) are secreted from the follicles in increasing amounts. In the human cycle, high blood levels of estradiol, somewhere around day 12-16 of the cycle, stimulate a large secretion of LH, which produces ovulation. At this point in time, the woman's basal body temperature (BBT) falls to its lowest point during the cycle. After ovulation (expulsion of ovum from ovary into oviduct), the follicle cavity undergoes cellular changes and is transformed into a body called a corpus luteum. This corpus futeum principally secretes another ovarian hormone, progesterone. This hormone, together with earlier stimulation by estrogen, prepares the lining of the uterus (endometrium) for acceptance of the fertilized ovum (implantation), if the woman has become pregnant. These same hormones"feed back"to inhibit FSH and LH secretion if the woman is pregnant, therefore preventing further ovulation until the pregnancy has been completed. See Buhrow. et al., J. Biol. Chem. , 258: 7824-7826 (1983).

Much research has been done to develop methods to control follicular development and subsequent ovulation. Such methods would either induce maturation in the case of patients with compromised ovulation or conversely the

method would seek to inhibit ovulation as a means of fertility control. Currently infertility in humans ranges from approximately 10-15% of couples and the risk of infertility is doubled for women between the ages of 35-44 as compared to women between the ages of 30-34. In the United States, infertility can, in certain instances, be accounted for by problems in the female.

Currently, biochemical female reproductive control is primarily accomplished through regimens of hormones such as estrogen and progesterone. Although this method of birth control is quite effective in preventing pregnancy, there are many side effects to the administration of additional hormones. Often such forms of birth control cause emotional and physiological disturbances, resulting in mood shifts or loss of libido. Health risks such as stroke and heart problems and cancer incidence increase for women who smoke while using hormonal birth control. In addition, these forms of fertility control are only potent when taken on a regularly basis. It is advisable to discontinue administration of hormones should fertilization occur in order to diminish chances of health complications.

There is a continued need in the field for new methods to modulate ovulation.

The present invention addresses this need.

Relevant Literature U. S. Patents of interest include : 6,472, 374; 5,968, 780. Published U. S.

Applications of interest include : 20030027998; 20030022279. Additional references of interest include : Dekel & Sherizly, Endocrinology (1985) 116: 406; Eppig, Seminars in Developmental Biology (1994) 5: 51; Espey & Richards, Biol Reprod (2002) 67: 1662-70; Hewitt & Korach, Steroids (2000) 65: 551-7; Prochazka et al., Mol. Reprod. Dev. (2000) 56: 63; and Goud et al., Hum. Reprod. (1998) 13: 1638.

SUMMARY OF THE INVENTION Methods and compositions for modulating ovulation are provided. In practicing the subject methods, an effective amount of at least one modulator of at least one epidermal growth factor (EGF)-related growth factor is administered to the female subject to modulate ovulation in the female subject. Also provided are compositions, kits, and systems for use in practicing the same.

One feature of the invention provides methods for modulating ovulation by administering to a subject an effective amount of at least one modulator of at least

one EGF-related growth factor activity. Such modulation could be in the form of either enhancing or inhibiting ovulation. In such a method, the EGF-related growth factor is chosen from amphiregulin (AR), epiregulin (EPI), and betacellulin (BTC). In certain embodiments, the modulation affects the activity of at least two EGF-related growth factors, and in certain embodiments, the modulation affects the activity of three EGF-related growth factors. The modulator used in such a method may comprise of polypeptides, nucleic acids, small molecules, or any combination thereof.

Another feature of the invention provides compositions comprising of an effective amount of at least one modulator of at least two EGF-related growth factor activities in conjunction with a pharmaceutical acceptable vehicle. Such a modulator may comprise of polypeptides, nucleic acids, small molecules, or any combination thereof. The composition may comprise of at least two distinct modulators of EGF-related growth factor activities, or it may comprise of at least three distinct modulators of EGF-related growth factor activities. Such EGF-related growth activities are chosen from AR, EPI, and BTC.

Yet another feature of the invention provides kits and systems which consist of at least one modulator of at least one EGF-related growth factor and instructions for using the modulator to affect ovulation in the subject. The kit or systems may comprise of two distinct modulators of EGF-related growth factor activity, or three distinct modulators of EGF-related growth factor activity. Such EGF-related growth factor activities used in the kit or system are chosen from AR, EPI, and BTC. The modulator included in the kit or system may comprise of polypeptides, nucleic acids, small molecules, or any combination thereof.

Also provided are methods of in vitro maturation. In such methods, follicular/oocyte cultures are treated with an effective amount of at least one modulator of at least one epidermal growth factor (EGF)-related growth factor, where the cultures may be treated with one or more additional factors, as desired, <BR> <BR> e. g. , HCG, etc. In these embodiments, the methods are analogous to known in vitro maturation methods, with the exception that, the cells/cultures thereof are also treated with an effective amount of at least one modulator of at least one epidermal growth factor (EGF)-related growth factor.

These and other objects, advantages, and features of the invention will become apparent to those persons skilled in the art upon reading the details of the invention as more fully described below.

BRIEF DESCRIPTION OF THE DRAWINGS The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to-scale.

On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures: FIGS. 1A-C illustrate the in vivo regulation of AP, EPI, and BTC expression by luteinizing hormone-human choriogonadotropic hormone (LH/hCG).

FIG. 1A is chip hybridization analysis illustrating the level of messenger RNA expression of members of the EGF growth factor family at different times after hCG stimulation in vivo. Each point is the mean SE of three determinations.

FIG. 1 B is a northern blot analysis of AR, EPI, and BTC mRNA expression in the ovary at different times after hCG stimulation in vivo. Total RNA was prepared and hybridized to probes corresponding to EPI, BTC and AR. The staining of the 28S RNA was used as a control for the loading.

FIG. 1C is an in situ hybridization of AR, EPI, and BTC mRNA expression in ovaries of immature mice primed with pregnant mare's serum gonadotropin (PMSG) for 44 h (0 time) and 3h after hCG injection.

FIGS. 2A-B illustrate fluorescence-activated cell sorting (FACS) of EPI expression in granulosa cells after in vivo hCG stimulation.

FIG. 2A is a FACS analysis of EPI expression where granulosa cells were harvested from ovaries of mice 0-4 hours after injection with hCG. Cells were labeled and analyzed by FACS using an EPI-specific antibody or with control IgGs.

FIG 2B is graph illustrating the percent of cells that become positive for EPI after injection with hCG.

FIGS. 3A-D illustrate that EGF-related growth factors mimic the LH effects on the follicle.

FIG. 3A is a graph illustrating the induction of meiotic maturation by AR, EPI, and BTC. Preovulatory follicles (POFs) were incubated for 4 h in the presence of

LH (1 Fg/ml), or increasing concentrations of AR, EPI, and BTC. Resumption of meiosis was measured by scoring the germinal vesicle breakdown (GVBD) in oocytes isolated from the follicles.

FIG. 3B is a graph illustrating the time course of AR, EPI, and LH stimulation of meiotic resumption. POFs were incubated with AR or EPI (100 nM) or LH (1 , ug/ml). At the time indicated in the abscissa, oocytes were retrieved from the follicles and scored for GVBD.

FIG. 3C illustrates images of an organ culture showing expansion of cumulus-oocyte complexes (COC) exposed for 8-12 h in MEM, MEM supplemented with 5% serum, FSH, AR, EPI, or BTC. Similar results were obtained using POFs.

FIG. 3D is a northern blot of semi-quantitative RT-PCR analysis of LH, EPI, AR, and BTC induction of genes involved in cumulus expansion. Follicles were cultured for 3 h with LH (1 lig/ml) or with AR, BTC, or EPI (100 nM). At the end of the culture, mRNA was extracted and analyzed by RT-PCR with primers specific for Cox2, Has2, Tsg6, and GAPDH.

FIGS. 4A-B demonstrate that EGF receptor (EGFR) phosphorylation is required for LH action.

FIG. 4A illustrates the effect of hCG injection on EGFR phosphorylation.

Ovaries were collected at different times after hCG injection, homogenized in RIPA buffer, and supernatants were immunoprecipitated with anti-EGFR antibody.

Immunoprecipitated samples were separated by SDS-PAGE and blotted with either a phospho-tyrosine antibody or an EGFR-specific antibody. The ratio between the intensity of the bands is reported as a bar graph as the mean SE of the three experiments performed.

FIG. 4B, panels A and B illustrate the effect of incubation of follicles with LH and increasing concentrations of the EGFR tyrosine kinase-selective inhibitors C56 (open circles) and AG1478 (filled circles). Incubation was terminated after four hours for GVBD (POF) or twelve hours for cumulus expansion. The effect of the inhibitors on spontaneous maturation was determined using COCs.

FIG. 4B, panel C is a northern blot of semi-quantitative RT-PCR analysis of the induction of the AP mRNA in follicles incubated for one hour with either the control, LH (1 lig/ml), or LH in the presence of AG1478 (3 I1M) or C56 (1 uM).

FIG. 5 is an in situ hybridization of expression of AR, EPI, and BTC in wild type and PDE4D-/-ovary three hours after hCG injection. EGF-related growth factor mRNA is absent in most of the large follicles of the PDE4D-/-ovary. The expression of these mRNAs was restricted to the few follicles that appear microscopically normal. The few oocytes ovulated by the PDE4D-/-mice are consistent with the presence of these luteinizing, unruptured follicles.

FIG. 6 is a graph illustrating that AG1478 concentration depends on inhibition of EPI and LH induced GVBD. Preovulatory follicles were incubated with increasing concentrations of AG1478 in the presence of either LH (1pg/ml) or EPI (100 nM). At the end of the 4 h incubation, oocytes were dissected free of granulosa cells and scored for GVBD. At least 20 follicles for each point were used.

FIG. 7 shows LH-induced expression of rER, rAR and rBTC mRNAs in preovulatory follicles in vitro. Panel A is a gel showing expression of the 3 genes examined and S16 as standard; Panel B is a summary of the results. Mean SEM are shown. LH-treated follicles differ significantly from untreated ones cultured for the same time; (b: p<0.0001 ; c: p< 0.002 for rER and rBTC and p< 0.03 for rAR) as revealed by one-way ANOVA-analysis.

FIG. 8 shows resumption of meiosis in rat follicle-enclosed oocytes in response to EGF-like growth factors. Panel A shows induction of meiosis by EGF- like ligands. Panel B shows inhibition of LH-induced maturation by AG1478.

Preovulatory follicles were cultured for 24 h with EGF-like factors, LH or LH+AG1478 (10 uM) as indicated. The number of examined oocytes is indicated on columns. The bars represent the percentage of GVB (mean SEM) of at least 3 groups of oocytes in two independent experiments (excluding 10 nM BTC with only 2 replicates, not included in statistical analysis) (b vs. control p< 0.03 ; c vs. control p<0.0002 ; d vs. control p<0.0001 ; e vs. LH p<0. 0001).

FIG. 9 shows EGFR phosphorylation and induction of resumption of meiosis by LH. Follicles were either untreated or incubated with LH (1 pg/ml), LH+AG43 (10 uM) and LH+AG 1478 (10 pM) (lanes 1,2, 3, and 4, respectively in A) for 6 hr followed by immunoblotting with the EGFR phophospecific PY1068 antibody and, after stripping by Ponceau Red for 2 hours, with EGFR specific antibody. In two separate experiments, each including 8 rats, 39 follicles were cultured for each treatment variable. Total protein extracted was applied to each lane. Panel A shows

a representative blot. Panel B shows a summary of two repeat experiments. The phosphorylated EGFR immunoblotting for each lane was standardized by EGFR.

The bars show the ratios for each variable as related to the mean of AG43, which is determined as 100%. The dots represent the values of two independent experiments. Panel C shows resumption of meiosis in rat follicle-enclosed oocytes.

Preovulatory follicles were cultured for 6 h with indicated additives (LH-g/ml ; AGs-10pM). The number of oocytes examined is indicated on columns. The bars represent the percentage of mature oocytes +/-SEM.

FIG. 10 shows the effect of AG1478 on ovulation. The vehicle (control) or the drug were injected unilaterally to the periovarian sac (bursa). The number of rats in each group are represented on the columns; the bars indicate the mean nSEM. * p< 0.02 AG1478 treated vs. vehicle treated control and p<0.0005 vs. untreated contralateral ovary as obtained by student t-test analysis.

FIG. 11 shows histological appearance of control (Panel A) and AG1478 treated ovaries (Panels B-E). Panel B shows follicles with entrapped oocytes: one at metaphase (black arrow) and the second with GV (white arrow); Panel C shows mmature oocyte with intact GV; Panel D shows mature oocyte at metaphase 11 ; Panel E shows mature oocyte with first polar body. Each bar-100 pm.

FIG. 12 shows inhibition of meiotic maturation and ovulation by AG1478 administration in vivo. Unruptured large antral follicles were counted and the meiotic status of entrapped oocytes defined in serial sections of AG1478-treated and their control, untreated contralateral ovaries. The data are presented as the percentage of large antral follicles with oocytes and the percentage of mature oocytes entrapped in them. The number of counted follicles and oocytes on columns.

FIG. 13 shows LH and ER stimulation of rat HAS-2, COX-2, TSG-6 mRNA expression in preovulatory follicles in vitro. Preovulatory follicles were explanted and cultured for different periods as described. A representative gel is shown (Panel A) and the cumulative results of 3 independent experiments (Panel B).

FIG. 14 shows inhibition of LH-induced expression of rHAS-2, rCOX-2 and rTSG-6 mRNA by AG1478. Preovulatory follicles were explanted and cultured for 6 h. A representative gel is shown (Panel A) and the cumulative results of 3 independent experiments (Panel B).

FIG. 15 shows ovarian expression in vivo of rHAS-2, rCOX-2 and rTSG-6 mRNA in AG1478 or vehicle-injected rats. The inhibitor or vehicle was injected unilaterally into the ovarian bursa (50 pi ; grey columns) and hCG was administered IP 30 minutes afterwards. The contralateral ovaries served as controls (black columns). The rats were sacrificed 6 h after hCG-stimulation of ovulation and the ovaries were processed for semiquantitative RT-PCR. Treatments with different letters differ significantly as revealed by one-way ANOVA-analysis (AG1478 injected vs. vehicle injected).

FIG. 16 shows the effect of Galardin on LH or ER-induced maturation of rat follicle-enclosed oocytes. The broad-spectrum MMP inhibitor Galardin (20 pM) was added 30 minutes prior to ER or LH. Treatments with different letters differ significantly (p<0.001) as revealed by one-way ANOVA-analysis.

DETAILED DESCRIPTION OF THE INVENTION Methods and compositions for modulating ovulation are provided. In practicing the subject methods, an effective amount of at least one modulator of at least one EGF-related growth factor is administered to the female subject to modulate ovulation in the female subject. Also provided are compositions, kits, and systems for use in practicing the same.

Before the present invention is described, it is to be understood that this invention is not limited to the particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also

encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the exemplary methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

It must be noted that as used herein and in the appended claims, the singular forms"a,""an,"and"the"include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to"a modulator"includes a plurality of such modulators and reference to"the EGF-related growth factor"includes reference to one or more EGF-related growth factor and equivalents thereof known to those skilled in the art, and so forth.

It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as"solely,""only"and the like in connection with the recitation of claim elements, or use of a"negative"limitation.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

In further describing the subject invention, the methods will be described first, followed by a review of representative applications in which the methods find use, as well as a review of pharmaceutical compositions and kits and systems thereof that find use in practicing the subject methods.

Methods As summarized above, the subject invention provides methods and compositions for modulating ovulation, specifically ovulation in a female subject. By ovulation is meant the production of ova, which may include the discharge of eggs from the ovary. The term modulation includes both enhancing and decreasing (inhibiting) ovulation. Accordingly, modulation may be in the form of either enhancing the ovulation of the female subject, or it may be in the form of inhibiting the ovulation of the female subject. By enhancing ovulation is meant at least increasing the rate of ovulation in a female subject, at least about 2-fold, such as at least about 5-fold, including at least about 10-fold, or where ovulation is non- existent in the subject, enhancing means at least causing at least one ovulation event in the subject. By inhibiting ovulation is meant at least decreasing the rate of ovulation in a female subject, by at least about 2-fold, such as at least about 5-fold, including at least about 10-fold, or substantially if not completely stopping ovulation, such that the subject ceases to ovulate.

The subject methods may be used to modulate ovulation in a variety of different types of subjects or hosts. Generally the subjects or hosts are"mammals" or"mammalian,"where these terms are used broadly to describe organisms which <BR> <BR> are within the class mammalia, including the orders carnivore (e. g. , dogs and cats),<BR> rodentia (e. g. , mice, guinea pigs, and rats), and primates (e. g. , humans, chimpanzees, and monkeys). In many embodiments, the subjects will be humans.

Ovulation is modulated in a subject according to the subject methods by administering to the subject at least one modulator (i. e., modulating agent) of at least one EGF-related growth factor activity. By"EGF-related growth factor activity" is meant the activity of an EGF-related growth factor. The term"EGF-related growth factor"refers to growth factors that are members of the epidermal growth factor (EGF) family of growth of factors, where in many embodiments the specific EGF related growth factors of interest are betacellulin, amphiregulin, and epiregulin. In many embodiments, ovulation is modulated by modulating the activity of at least one of betacellulin, amphiregulin, and epiregulin in the subject. Modulating the activity includes both increasing and decreasing the activity of the target EGF-related growth factor (s) in the host.

Where the activity of the target growth factor is increased, the activity of the growth factor is increased in many embodiments by at least about 2-fold, such as at least about 5-fold, including at least about 10-fold, as compared to a control.

Alternatively, in cases where activity of the target growth factor is so low that it is undetectable, activity of the target growth factor is considered to be enhanced if activity is increased to a level that is easily detectable.

Conversely, where the activity of the target growth factor is decreased, the activity of the growth factor is decreased in many embodiments by at least about 2- fold, such as at least about 5-fold, including at least about 10-fold, as compared to a control.

As indicated above, the activity of at least one EGF-related growth factor is modulated. In certain embodiments, the activity of only one EGF-related growth factor is modulated, e. g. , one of betacellulin, amphiregulin, and epiregulin. In certain<BR> embodiments, the activity of two EGF-related growth factors is modulated, e. g. , two of betacellulin, amphiregulin, and epiregulin. In certain embodiments, the activity of three EGF-related growth factors is modulated, e. g., all three of betacellulin, amphiregulin, and epiregulin.

In practicing the subject methods, the activity of the target EGF-related growth factor activity (s) is modulated by administering to the subject an effective amount of at least one modulating agent. By"effective amount"is meant an amount which is sufficient to effect the desired modulation in growth factor activity/ovulation, as defined herein, when administered to the subject. The effective amount will vary depending on the subject and the particular modulation desired, the severity of the condition being treated and the manner of administration, and may be determined routinely by one of ordinary skill in the art.

In certain embodiments, a single modulatory agent that modulates the activity of at least one, and sometimes two or more, including three or more, EGF-related growth factors may be employed. In yet other embodiments, a single modulator agent for each target growth factor is employed, such that where the methods modulate two different growth factors, two different modulator agents are administered to the subject. Likewise, where the methods modulate three different growth factors, three different modulator agents are administered to the subject.

As indicated above, the modulator agent (s) may be enhancers or inhibitors of the target growth factor activity (s). The active agent may be one or a mixture of a <BR> <BR> variety of different compounds, including: polynucleotide compositions (e. g. , coding sequences, antisense compositions, siRNA compositions, etc.), polypeptide, including antibody, compositions, naturally occurring or synthetic small molecule compounds, etc.

In certain embodiments, the active agents administered to the host are polynucleotide or nucleic acid compositions. The nucleic acids may be coding <BR> <BR> sequences, e. g. , genes, gene fragments etc. , which may be present in expression vectors, where such vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences.

Transcription cassettes may be prepared that include a transcription initiation region, the target gene or fragment thereof, and a transcriptional termination region.

The transcription cassettes may be introduced into a variety of vectors, e. g. plasmid ; retrovirus, e. g. lentivirus ; adenovirus; and the like, where the vectors are able to transiently or stably be maintained in the cells, usually for a period of at least about one day, more usually for a period of at least about several days to several weeks.

The nucleic acid coding sequence (mRNA) of human amphiregulin is provided a NM_001432, D30783. 1 t ; the nucleic acid coding sequence (mRNA) human epiregulin is provided at NM_001432, D30783.1 and the nucleic acid coding sequence (mRNA) of human betacellulin is provided at NM 001729, BC011618. 2.

The nucleic acids may be human nucleic acids or homologs or nucleic acids (or fragments thereof) from other species, i. e., other animal species, where such homologs or proteins may be from a variety of different types of species, usually <BR> <BR> mammals, e. g. , rodents, such as mice, rats; domestic animals, e. g. horse, cow, dog,<BR> cat; and primates, e. g. , monkeys, baboons, humans etc. By homolog is meant a nucleic acid having at least about 35%, usually at least about 40% and more usually at least about 60% sequence identity to the specific human nucleic acids as identified above, where sequence identity is as measured by the BLAST compare two sequences program available on the NCBI website using default settings.

In yet other embodiments of the invention, the active agent is an agent that modulates, and generally decreases or down regulates, the expression of the target growth factor (s) in the host. Antisense molecules can be used to down-regulate

expression of a gene in cells. The anti-sense reagent may be antisense oligonucleotides (ODN), particularly synthetic ODN having chemical modifications from native nucleic acids, or nucleic acid constructs that express such anti-sense molecules as RNA. The antisense sequence is complementary to the mRNA of the targeted gene, and inhibits expression of the targeted gene products. Antisense molecules inhibit gene expression through various mechanisms, e. g. by reducing the amount of mRNA available for translation, through activation of RNAse H, or steric hindrance. One or a combination of antisense molecules may be administered, where a combination may comprise multiple different sequences.

Antisense molecules may be produced by expression of all or a part of the target gene sequence in an appropriate vector, where the transcriptional initiation is oriented such that an antisense strand is produced as an RNA molecule.

Alternatively, the antisense molecule is a synthetic oligonucleotide. Antisense oligonucleotides will generally be at least about 7, usually at least about 12, more usually at least about 20 nucleotides in length, and not more than about 500, usually not more than about 50, more usually not more than about 35 nucleotides in length, where the length is governed by efficiency of inhibition, specificity, including absence of cross-reactivity, and the like. It has been found that short oligonucleotides, of from 7 to 8 bases in length, can be strong and selective inhibitors of gene expression (see Wagner et a/. (1996), Nature Biotechnol. 14: 840- 844).

A specific region or regions of the endogenous sense strand mRNA sequence is chosen to be complemented by the antisense sequence. Selection of a specific sequence for the oligonucleotide may use an empirical method, where several candidate sequences are assayed for inhibition of expression of the target gene in an in vitro or animal model. A combination of sequences may also be used, where several regions of the mRNA sequence are selected for antisense complementation.

Antisense oligonucleotides may be chemically synthesized by methods known in the art. Suitable oligonucleotides are chemically modified from the native phosphodiester structure, in order to increase their intracellular stability and binding affinity. A number of such modifications have been described in the literature, which alter the chemistry of the backbone, sugars or heterocyclic bases.

Among useful changes in the backbone chemistry are phosphorothioates; phosphorodithioates, where both of the non-bridging oxygens are substituted with sulfur ; phosphoroamidites; alkyl phosphotriesters and boranophosphates. Achiral phosphate derivatives include 3'-0'-5'-S-phosphorothioate, 3'-S-5'-0- phosphorothioate, 3'-CH2-5'-O-phosphonate and 3'-NH-5'-0-phosphoroamidate.

Peptide nucleic acids replace the entire ribose phosphodiester backbone with a peptide linkage. Sugar modifications are also used to enhance stability and affinity.

The a-anomer of deoxyribose may be used, where the base is inverted with respect to the natural p-anomer. The 2'-OH of the ribose sugar may be altered to form 2'-0- methyl or 2'-0-allyl sugars, which provides resistance to degradation without comprising affinity. Modification of the heterocyclic bases must maintain proper base pairing. Some useful substitutions include deoxyuridine for deoxythymidine; 5-methyl-2'-deoxycytidine and 5-bromo-2'-deoxycytidine for deoxycytidine. 5- propynyl-2'-deoxyuridine and 5-propynyl-2'-deoxycytidine have been shown to increase affinity and biological activity when substituted for deoxythymidine and deoxycytidine, respectively.

As an alternative to anti-sense inhibitors, catalytic nucleic acid compounds, e. g. ribozymes, anti-sense conjugates, etc. may be used to inhibit gene expression.

Ribozymes may be synthesized in vitro and administered to the patient, or may be encoded on an expression vector, from which the ribozyme is synthesized in the targeted cell (for example, see International patent application WO 9523225, and Beigelman et al. (1995), Nucl. Acids Res. 23: 4434-42). Examples of oligonucleotides with catalytic activity are described in WO 9506764. Conjugates of anti-sense ODN with a metal complex, e. g. terpyridylCu (II), capable of mediating mRNA hydrolysis are described in Bashkin et al. (1995), Appl. Biochem. Biotechnol.

54: 43-56.

Alternatively, gene expression can be modified by gene silencing using double-strand RNA (Sharp (1999) Genes and Development 13 : 139-141). RNAi, otherwise known as double-stranded RNA interference (dsRNAi) or small interfering RNA (siRNA), has been extensively documented in the nematode C. elegans (Fire, A. , et al, Nature, 391,806-811, 1998) and an identical phenomenon occurs in plants, in which it is usually referred to as post-transcriptional gene silencing (PTGS) (Van Blokland, R. , et al., Plant J., 6: 861-877,1994 ; deCarvalho-Niebel, F. , et al.,

Plant Cell, 7: 347-358,1995 ; Jacobs, J. J. M. R. et al., Plant J., 12: 885-893,1997 ; reviewed in Vaucheret, H. , et al.., Plant J., 16: 651-659,1998). The phenomenon also occurs in fungi (Romano, N. and Masino, G., Mol. Microbiol., 6: 3343-3353, 1992, Cogoni, C. , et al., EMBO J. , 15: 3153-3163; Cogoni, C. and Masino, G., Nature, 399: 166-169,1999), in which it is often referred to as"quelling". RNAi silencing can be induced many ways in plants, where a nucleic acid encoding an RNA that forms a"hairpin"structure is employed in most embodiments. Alternative strategies include expressing RNA from each end of the encoding nucleic acid, making two RNA molecules that will hybridize. Current strategies for RNAi induced silencing in plants are reviewed by Carthew et al (Curr Opin Cell Biol. 2001 13: 244- 8). RNAi is also described in WO 02/44321 and WO 01/68836; the priority documents of which are herein incorporated by reference.

Also of interest are polypeptides, e. g. , proteinaceous, active agents. Specific polypeptide agents include proteins or active fragments thereof, e. g., EGF-related growth factor proteins (such as amphiregulin, epiregulin, and betacellulin), etc. The amino acid sequence of human amphiregulin is provided at NP001648, A34702; the amino acid sequence of human epiregulin is provided at NP001423 and the amino acid sequence of human betacellulin is provided at NP001720, JC146. The proteins may be human proteins or homologs or proteins (or fragments thereof) from other species, i. e. , other animal species, where such homologs or proteins may be from a variety of different types of species, usually mammals, e. g., rodents, such as mice, rats; domestic animals, e. g. horse, cow, dog, cat; and primates, e. g. , monkeys, baboons, humans etc. By homolog is meant a protein having at least about 35%, usually at least about 40% and more usually at least about 60% amino acid sequence identity to the specific human proteins as identified above, where sequence identity is as measured by the BLAST compare two sequences program available on the NCBI website using default settings.

Another specific type of polypeptide active agent of interest is an antibody agent that modulates the target growth factor activity (s) in the host. The antibodies may be monoclonal or polyclonal, and produced according to methods known in the art. Antibody fragments, such as Fv, F (ab') 2 and Fab may be prepared by cleavage of the intact protein, e. g. by protease or chemical cleavage.

Alternatively, a truncated gene is designed. For example, a chimeric gene encoding a portion of the F (ab') 2 fragment would include DNA sequences encoding the CH1 domain and hinge region of the H chain, followed by a translational stop codon to yield the truncated molecule. Consensus sequences of H and L J regions may be used to design oligonucleotides for use as primers to introduce useful restriction sites into the J region for subsequent linkage of V region segments to human C region segments. C region cDNA can be modified by site directed mutagenesis to place a restriction site at the analogous position in the human sequence.

Expression vectors include plasmids, retroviruses, YACs, EBV derived episomes, and the like. A convenient vector is one that encodes a functionally complete human CH or CL immunoglobulin sequence, with appropriate restriction sites engineered so that any VH or VL sequence can be easily inserted and expressed. In such vectors, splicing usually occurs between the splice donor site in the inserted J region and the splice acceptor site preceding the human C region, and also at the splice regions that occur within the human CH exons.

Polyadenylation and transcription termination occur at native chromosomal sites downstream of the coding regions. The resulting chimeric antibody may be joined to any strong promoter, including retroviral LTRs, e. g. SV-40 early promoter, (Okayama et al. (1983) Mol. Ce//. Bio. 3: 280), Rous sarcoma virus LTR (Gorman et al. (1982) P. N. A. S. 79: 6777), and moloney murine leukemia virus LTR (Grosschedl et al. (1985) Cell41 : 885) ; native Ig promoters, etc.

Also of interest as modulator agents are naturally occurring or synthetic small molecule compounds, which include numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 50 and less than about 2,500 daltons. Candidate agents include functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups.

The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules

including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs, or combinations thereof.

In practicing the subject methods, the active agent or agents are typically <BR> <BR> administered to the host in a physiologically acceptable delivery vehicle, e. g. , as a pharmaceutical preparation. A variety of representative formulations, dosages, routes of administration for candidate agents, nucleic acid delivery vehicles, and nucleic acid formulations for nucleic acid delivery are described below.

The one or more active agents employed in the subject methods are typically administered to the subject to achieve the desired modulation in activity/ovulation.

The active agents may be administered in convenient formulations, including pharmaceutical formulations that include at least one agent which modulates at least one target EGF-related growth factor activity in the subject. In general, a formulation comprises an effective amount of at least one modulator agent as described above. An"effective amount"refers to an amount that is sufficient to produce a desired result, e. g., increase or decrease ovulation rate.

In the subject methods, the active agent (s) may be administered to the host using any convenient means.

Thus, the agent can be incorporated into a variety of formulations for therapeutic administration. More particularly, the agents of the present invention can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutical acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, and aerosols.

In pharmaceutical dosage forms, the agents may be administered in the form of their pharmaceutically acceptable salts, or they may also be used alone or in appropriate association, as well as in combination, with other pharmaceutically active compounds. The following methods and excipients are merely exemplary and are in no way limiting.

For oral preparations, the agents can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, corn starch, or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch,

or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose ; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives, and flavoring agents.

The agents can be formulated into preparations for injection by dissolving, suspending, or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids, or propylene glycol ; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers, and preservatives.

The agents can be utilized in aerosol formulation to be administered via inhalation. The compounds of the present invention can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like.

Furthermore, the agents can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases. The compounds of the present invention can be administered rectally via a suppository. The suppository can include vehicles such as cocoa butter, carbowaxes, and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature.

Unit dosage forms for oral or rectal administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet, or suppository, contains a predetermined amount of the composition containing one or more inhibitors. Similarly, unit dosage forms for injection or intravenous administration may comprise the inhibitor (s) in a composition as a solution in sterile water, normal saline, or another pharmaceutical acceptable carrier.

The term"unit dosage form, "as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of compounds of the present invention calculated in an amount sufficient to produce the desired effect in association with a pharmaceutical acceptable diluent, carrier or vehicle. The specifications for the novel unit dosage forms of the present invention depend on the particular

compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host.

Other modes of administration will also find use with the subject invention.

For instance, an agent of the invention can be formulated in suppositories and, in some cases, aerosol and intranasal compositions. For suppositories, the vehicle composition will include traditional binders and carriers such as, polyalkylene glycols, or triglycerides.. Such suppositories may be formed from mixtures containing the active ingredient in the range of about 0.5% to about 10% (w/w), preferably about 1 % to about 2%.

Intranasal formulations will usually include vehicles that neither cause irritation to the nasal mucosa nor significantly disturb ciliary function. Diluents such as water, aqueous saline or other known substances can be employed with the subject invention. The nasal formulations may also contain preservatives such as, but not limited to, chlorobutanol and benzalkonium chloride. A surfactant may be present to enhance absorption of the subject proteins by the nasal mucosa.

An agent of the invention can be administered as injectables. Typically, injectable compositions are prepared as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. The preparation may also be emulsified or the active ingredient encapsulated in liposome vehicles.

Suitable excipient vehicles are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, if desired, the vehicle may contain minor amounts of auxiliary substances such as wetting or emulsifying agents or pH buffering agents. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art. See, e. g. , Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, 17th edition, 1985; Remington: The Science and Practice of Pharmacy, A. R. Gennaro, (2000) Lippincott, Williams & Wilkins. The composition or formulation to be administered will, in any event, contain a quantity of the agent adequate to achieve the desired state in the subject being treated.

The pharmaceutical acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are readily available to the public. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity

adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.

Although the dosage used will vary depending on the clinical goals to be achieved, a suitable dosage range is one which provides up to about 1 ug to about 1, 000 ug or about 10,000 pg of an agent that modulates the target EGF-related growth factor activity. Those of skill will readily appreciate that dose levels can vary as a function of the specific compound, the severity of the symptoms and the susceptibility of the subject to side effects. Suitable dosages for a given compound are readily determinable by those of skill in the art by a variety of means.

Conventional and pharmaceutically acceptable routes of administration include intranasal, intramuscular, intratracheal, intratumoral, subcutaneous, intradermal, topical application, intravenous, rectal, nasal, oral and, other parenteral routes of administration. Routes of administration may be combined, if desired, or adjusted depending upon the agent and/or the desired effect. The composition can be administered in a single dose or in multiple doses.

The agent can be administered to a host using any available conventional methods and routes suitable for delivery of conventional drugs, including systemic or localized routes. In general, routes of administration contemplated by the invention include, but are not necessarily limited to, enteral, parenteral, or inhalational routes.

Parenteral routes of administration other than inhalation administration include, but are not necessarily limited to, topical, transdermal, subcutaneous, intramuscular, intraorbital, intracapsular,. intraspinal, intrasternal, and intravenous routes, i. e., any route of administration other than through the alimentary canal.

Parenteral administration can be carried to effect systemic or local delivery of the agent. Where systemic delivery is desired, administration typically involves invasive or systemically absorbed topical or mucosal administration of pharmaceutical preparations.

The agent can also be delivered to the subject by enteral administration.

Enteral routes of administration include, but are not necessarily limited to, oral and rectal (e. g. , using a suppository) delivery.

Methods of administration of the agent through the skin or mucosa include, but are not necessarily limited to, topical application of a suitable pharmaceutical

preparation, transdermal transmission, injection and epidermal administration. For transdermal transmission, absorption promoters or iontophoresis are suitable methods. lontophoretic transmission may be accomplished using commercially available"patches"which deliver their product continuously via electric pulses through unbroken skin for periods of several days or more.

A subject polynucleotide modulator agent can be delivered as a naked <BR> <BR> polynucleotide, or associated with (complexed with) a delivery vehicle. "Associated<BR> with,"or"complexed with, "encompasses both covalent and non-covalent interaction of a polynucleotide with a given delivery vehicle. In certain embodiment, an agent is a nucleic acid. Nucleic acids may be delivered using several different vehicles, including viral and non-viral delivery vehicles.

A subject polynucleotide can be associated with viral delivery vehicles. As used herein, a"viral delivery vehicle"intends that the polynucleotide to be delivered is encapsidated in a viral particle.

Numerous viral genomes useful in in vivo transformation and gene therapy are known in the art, or can be readily constructed given the skill and knowledge in the art. Included are replication competent, replication deficient, and replication conditional viruses. Viral vectors include adenovirus, mumps virus, a retrovirus, adeno-associated virus, herpes simplex virus (HSV), cytomegalovirus (CMV), vaccinia virus, and poliovirus, and non-replicative mutants/variants of the foregoing.

In some embodiments, a replication-deficient virus is capable of infecting slowly replicating and/or terminally differentiated cells, since the respiratory tract is primarily composed of these cell types. For example, adenovirus efficiently infects slowly replicating and/or terminally differentiated cells. In some embodiments, the viral genome itself, or a protein on the viral surface, is specific or substantially specific for cells of the targeted cell. A viral genome can be designed to be target cell-specific by inclusion of cell type-specific promoters and/or enhancers operably linked to a gene (s) essential for viral replication.

Where a replication-deficient virus is used as the viral genome, the production of virus particles containing either DNA or RNA corresponding to the polynucleotide of interest can be produced by introducing the viral construct into a recombinant cell line which provides the missing components essential for viral replication and/or production. Preferably, transformation of the recombinant cell line

with the recombinant viral genome will not result in production of replication- <BR> <BR> competent viruses, e. g. , by homologous recombination of the viral sequences of the recombinant cell line into the introduced viral genome. Methods for production of replication-deficient viral particles containing a nucleic acid of interest are well known in the art and are described in, for example, Rosenfeld et al., Science 252: 431-434,1991 ; Rosenfeld et al., Ce//68 : 143-155,1992 (adenovirus); U. S.

Patent No. 5,139, 941 (adeno-associated virus); U. S. Patent No. 4,861, 719 (retrovirus); and U. S. Patent No. 5,356, 806 (vaccinia virus). Methods and materials for manipulation of the mumps virus genome, characterization of mumps virus genes responsible for viral fusion and viral replication, and the structure and sequence of the mumps viral genome are described in Tanabayashi et al., J. Virol.

67: 2928-2931,1993 ; Takeuchi et al., Archiv. Virol., 128: 177-183,1993 ; Tanabayashi et al., Virol. 187: 801-804, 1992; Kawano et al., Virol., 179: 857-861, 1990; Elango et al., J. Gen. Virol. 69: 2893-28900,1988.

A subject polynucleotide can be administered using a non-viral delivery vehicle."Non-viral delivery vehicle" (also referred to herein as"non-viral vector") as used herein is meant to include chemical formulations containing naked or condensed polynucleotides (e. g, a formulation of polynucleotides and cationic <BR> <BR> compounds (e. g. , dextran sulfate)), and naked or condensed polynucleotides mixed with an adjuvant such as a viral particle (i. e., the polynucleotide of interest is not contained within the viral particle, but the transforming formulation is composed of <BR> <BR> both naked polynucleotides and viral particles (e. g. , adenovirus particles) (see, e. g.,<BR> Curiel et al. 1992 Am. J. Respir. Cell Mol. Biol. 6: 247-52) ). Thus"non-viral delivery vehicle"can include vectors composed of polynucleotides plus viral particles where the viral particles do not contain the polynucleotide of interest."Non-viral delivery vehicles"include bacterial plasmids, viral genomes or portions thereof, wherein the polynucleotide to be delivered is not encapsulated or contained within a viral particle, and constructs comprising portions of viral genomes and portions of bacterial plasmids and/or bacteriophages. The term also encompasses natural and synthetic polymers and co-polymers. The term further encompasses lipid-based vehicles. Lipid-based vehicles include cationic liposomes such as disclosed by Felgner et al (U. S. Patent Nos. 5,264, 618 and 5,459, 127; PNAS 84: 7413-7417, 1987; Annals N. Y. Acad. Sci. 772: 126-139,1995) ; they may also consist of neutral

or negatively charged phospholipids or mixtures thereof including artificial viral envelopes as disclosed by Schreier et al. (U. S. Patent Nos. 5,252, 348 and 5,766, 625).

Non-viral delivery vehicles include polymer-based carriers. Polymer-based carriers may include natural and synthetic polymers and co-polymers. Preferably, the polymers are biodegradable, or can be readily eliminated from the subject.

Naturally occurring polymers include polypeptides and polysaccharides. Synthetic polymers include, but are not limited to, polylysines, and polyethyleneimines (PEI) (see Boussif et al., PNAS 92: 7297-7301,1995) which molecules can also serve as condensing agents. These carriers may be dissolved, dispersed or suspended in a dispersion liquid such as water, ethanol, saline solutions and mixtures thereof. A wide variety of synthetic polymers are known in the art and can be used.

"Non-viral delivery vehicles"further include bacteria. The use of various bacteria as delivery vehicles for polynucleotides has been described. Any known bacterium can be used as a delivery vehicle, including, but not limited to non- pathogenic strains of Staphylococcus, Salmonella, and the like.

The polynucleotide to be delivered can be formulated as a DNA-or RNA-liposome complex formulation. Such complexes comprise a mixture of lipids which bind to genetic material (DNA or RNA) by means of cationic charge (electrostatic interaction). Cationic liposomes which may be used in the present invention include 3ß-[N-(N', N'-dimethyl-aminoethane)-carbamoyl]-cholesterol (DC-Chol), 1,2- bis (oleoyloxy-3-trimethylammonio-propane (DOTAP) (see, for example, WO 98/07408), lysinylphosphatidylethanolamine (L-PE), lipopolyamines such as lipospermine, N- (2-hydroxyethyl)-N, N-dimethyl-2, 3-bis (dodecyloxy)-1- propanaminium bromide, dimethyl dioctadecyl ammonium bromide (DDAB), dioleoylphosphatidyl ethanolamine (DOPE), dioleoylphosphatidyl choline (DOPC), N (1,2, 3-dioleyloxy) propyl-N, N, N-triethylammonium (DOTMA), DOSPA, DMRIE, GL- 67, GL-89, Lipofectin, and Lipofectamine (Thiery et al. (1997) Gene Ther. 4: 226- 237; Feigner et al., Annals N. Y. Acad. Sci. 772: 126-139,1995 ; Eastman et al., Hum.

Gene Ther. 8: 765-773,1997). Polynucleotide/lipid formulations described in U. S.

Patent No. 5,858, 784 can also be used in the methods described herein. Many of these lipids are commercially available from, for example, Boehringer-Mannheim, and Avanti Polar Lipids (Birmingham, AL). Also encompassed are the cationic

phospholipids found in U. S. Patent Nos. 5,264, 618,5, 223,263, and 5, 459, 127.

Other suitable phospholipids which may be used include phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingomyelin, phosphatidylinositol, and the like. Cholesterol may also be included.

Utility The subject methods find use in a variety of different applications in which modulation, e. g. , an increase or decrease, of ovulation is desired. As such, the subject methods find use in applications where at least a decrease, if not a complete cessation of ovulation is desired. Such applications include contraceptive applications, where the methods are practiced in order to prevent pregnancy in a subject.

Another embodiment is the use of the treatment of infertility in a subject, where at least an increase in the rate of ovulation in the subject is desired. By treatment is meant at least an amelioration of the symptoms associated with the infertility afflicting the host is achieved, where amelioration is used in a broad sense to refer to at least an increase in the rate of ovulation, where the increase may or may not be to the rate of ovulation that is considered normal for species of which the subject is a member. Infertility may also be treated according to the subject invention via in vitro maturation protocols.

A variety of hosts are tratable according to the subject methods. Generally such hosts are"mammals"or"mammalian,"where these terms are used broadly to describe organisms which are within the class mammalia, including the orders <BR> <BR> carnivore (e. g. , dogs and cats), rodentia (e. g., mice, guinea pigs, and rats), and<BR> primates (e. g. , humans, chimpanzees, and monkeys). In many embodiments, the hosts will be humans.

Pharmaceutical Formulations Also provided are pharmaceutical compositions containing at least one modulator of at least two different EGF-related growth factors, where the composition may include two or more different modulators, where the active agents- are present in a pharmaceutically acceptable delivery vehicle. Representative specific pharmaceutically acceptable delivery vehicles include those representative vehicles described above.

Kits & Systems Also provided are kits and systems that find use in practicing the subject methods, as described above. For example, kits and systems for practicing the subject methods may include one or more pharmaceutical formulations, which include one or more EGF-related growth factor modulator active agents, as described above. As such, in certain embodiments the kits may include a single pharmaceutical composition, present as one or more unit dosages, where the composition includes one or more active agents. In yet other embodiments, the kits may include two or more separate pharmaceutical compositions, each containing a separate active agent.

In addition to the above components, the subject kits may further include instructions for practicing the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit.

One form in which these instructions may be present is as printed information on a suitable medium or substrate, e. g. , a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, etc. Yet another means would be a computer readable medium, e. g. , diskette, CD, etc. , on which the information has been recorded. Yet another means that may be present is a website address which may be used via the internet to access the information at a removed site. Any convenient means may be present in the kits.

The term"system"as employed herein refers to a collection of active agents, present in a single composition or as disparate compositions that are brought together for the purpose of practicing the subject methods. For example, separately obtained dosage forms brought together and co-administered to a subject, according to the present invention, are a system according to the present invention.

The following examples are offered by way of illustration and not by way of limitation.

EXPERIMENTAL The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments

below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e. g. amounts, temperature, etc. ) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

Materials and Methods The following materials and methods were employed in Examples 1 to 4.

Animals All animal procedures were approved and followed guidelines of the Stanford University Administrative Panel on Laboratory Animal Care (A-PLAC). The PDE4D-'- mice were generated as previously described in Jin et al., (1999) P. N. A. S. 96: 1198.

Immature (20-30 day of age, body weight 10-12 g) PDE4D--female and heterozygous littermates, or C57BL wild type females were injected IP with 5 IU PMSG (Calbiochem, La Jolla, CA) to stimulate follicle development. After 46-48 h, animals were killed by cervical dislocation and ovaries excised. Group of mice were injected with an ovulatory dose of 5 IU hCG, and ovaries were excised at different times after the injection.

Analysis of the EGF Network by Hybridization to Immobilized Probes RNA was isolated from the PMSG-primed immature PDE4D-/-and heterozygous littermates at different times after hCG IP injection. Twenty to 40 tig of total RNA was processed for hybridization to immobilized oligonucleotides. The cDNA synthesis, in vitro transcription with Biotin labeled ribonucleotides and fragmentation to produce the oligonucleotide probes were performed following the instruction of the manufacturer (Affymetrix, Santa Clara, CA). The probes were hybridized to GeneChip murine genome U74A V. 2 array (Affymetrix, Santa Clara, CA). Data was analyzed using Affymetrix Microarray Suite 5.0 software, and GeneSpring 4.2 (Silicon Genetics, Redwood City, CA). To compare data from different chips, densitometric values were corrected for the intensity of the signal of 50% of the genes and data were expressed as intensity ratio. Each point is the mean SEM of three arrays. The significance of the difference of genes between PDE4D-/-and heterozygous type was analyzed by two-way ANOVA. The P values are: Epiregulin =0.0042, Amphiregulin =0.0005, Betacellulin = 0.02.

Northern Blot and Semi-Quantitative RT-PCR Total RNA from ovaries was isolated using TRlzol Reagent solution (Invitrogen Corp. , Carlsbad, CA). Twenty micrograms of total RNA were separated by electrophoresis and transferred to nylon membranes. The membranes were hybridized with 32P-labeled cDNA probes, washed and exposed using Kodak RX film at-80C.

For the semiquantitative measurements of gene expression, total RNA was extracted from preovulatory follicles. RT-PCR and Southern blot analysis was performed according to the procedure described previously (Park et at. , (2003), Mol.

Endo. 17: 117). Specific primers were used to amplify the following mRNAs; Cox2 (forward 5'-tgtacaagcagtggcaaagg-3' (SEQ ID NO.: 01), reverse 5'- gctgtggatcttgcacattg-3' (SEQ ID NO.: 02) ), Has2 (forward 5'- gcttgaccctgcctcatctgtgg-3' (SEQ ID NO.: 03), reverse 5'-ctggttcagccatctcagatatt-3' (SEQ ID NO.: 04) ), Tsg6, (forward, 5'-ttccatgtctgtgctgctggatgg-3' (SEQ ID NO.: 05),<BR> reverse 5'-agcctggatcatgttcaaggtcaaa-3' (SEQ ID NO.: 06) ), GAPDH (forward 5'- tgaaggtcggtgtgaacggatttggc-3' (SEQ ID NO.: 07), reverse 5'- catgtaggccatgaggtccaccac-3' (SEQ ID NO.: 08) ). For southern blot the following oligonucleotides were labeled with [32P]-yATP and T4 kinase (Invitrogen) : Cox2: 5'- tccagåtgctatctttgggg-3' (SEQ ID NO.: 09), Tsg-6 5'-ccacatgcaaaggagtgtgg-3' (SEQ ID NO.: 10); Has2: 5'-gctgtgtccagtgcataagc-3' (SEQ ID NO.: 11).

In Situ Hybridization Ovaries of PMSG or hCG treated mice were fixed in 4% paraformaldehyde (PFA) for 6 hours and incubated in 0.5M sucrose overnight at 4°C. The ovaries were embedded in OCT (Tissue-Tek), cut into 101lm sections, and mounted on Superfrost slides (Fisher, Pittsburgh, PA). Slides were post-fixed in PFA, treated with 0.2M HCI, 2x SSC at 70°C, pronase E (Sigma, St. Louis, MO), and 2mg/ml glycine. Slides were then subjected to an acetylation treatment and dehydrated in ethanol (30-100%). The AR, EPI and BTC cDNAs were subcloned in pBluescript vector and was linearized by EcoRl and BamHl, transcribed to synthesize [35S]- labeled RNA probes. Hybridization mixtures with antisense and sense RNA probes were added to the slide and incubated overnight at 50°C. Post-hybridization washes consisted of RNaseA treatment and decreasing concentration of SSC washes. Hybridized slides were then dehydrated and dried. Slides were dipped into

NTB2 Emulsion (Eastman Kodak, Rochester, NY), exposed for 2 days, and developed photographically, and then counter-stained with Gill's Hematoxylin and EosinY (0.5% w/v in ethanol). Following counterstaining, tissues were cleared with xylene, mounted with Permount, visualized and photographed with AxioCam (Zeiss).

FACS Analysis Granulosa cells were collected by follicle puncture at 0,2, 3 and 4 hours after hCG i. p. injection. Contaminating red blood cells were removed by erythrocyte lysing buffer (Sigma) and washed with Ca2+ Mg2+ free PBS containing 2% FBS (FACS buffer). Washed granulosa cells were incubated with mouse Fc-block (BD biosciences) for 15 min on ice followed by goat anti-Epi antibody (R&D systems) for 40 min on ice. After primary antibody incubation, the cells were washed with FACS buffer and incubated with fluorescein isothiocyanate (FITC)-conjugated swine anti- goat antibody (Caltag, Burlingame, CA) for 40 min on ice and washed with FACS buffer. As a control, cells were incubated with normal goat serum instead of the primary antibodies. The labeled cells were analyzed on a Becton Dickinson FACScalibur flow cytometer.

Preovulatory Follicle and Cumulus Oocyte Complex Cultures The preovulatory follicles (POF) and cumulus-oocyte complexes (COC) were isolated 46-48 h post injection (as described in Park et al., (2001) Endocrinology 142: 3828; Su et al., (2002) Endocrinology 143: 2221), and were cultured in MEM (Gibco, Grand Island, NY) medium supplemented with penicillin, streptomycin, and 5% of fetal bovine serum (Gemini, Woodland, CA). The preovulatory follicles or COC were cultured at 37 °C incubators in controlled atmosphere of 95% 02,5% C02, or 5% 02,5% C02,90% N2, respectively. LH (1 Lg/mi) EPI (100 nM) AR (300 nM) BTC (100 nM) (R&D system Minneapolis, MN) were added after 30 min pre-equilibration. At the end of the culture, the preovulatory follicles were punctured to release the cumulus oocyte complex. The oocyte maturation was assessed by scoring released oocytes for germinal vehicle breakdown (GVBD) after removal of cumulus cells. After culture for 12-15 h, COCs or COCs released from preovulatory follicles were scored for cumulus expansion as described (Vanderheyden et al., (1990) Dev. Bio. 140: 307). Cumuli at stage III and IV were considered fully

expanded. Each point reported in the figures is the mean SE of 50 or more POFs or COCs analyzed in two or three separate experiments.

AG1478 and compound 56 (Calbiochem, La Jolla, CA) were dissolved in DMSO and added to the POF 30 min prior to hormonal or growth factor stimulation.

Final concentration of DMSO in the culture was 0. 1 % Control follicles were incubated with the same concentration of vehicle.

Immnoprecipitation and Western Blot Analvsis Ovaries collected from gonadotropin-treated mice were homogenized in ice- cold RIPA buffer containing 10mM Na-orthovanadate, 50mM NaF and-1mM Na- pyrophosphate. After centrifugation at 14, 000xg for 5 min at 4C, supernatants were used for immunoprecipitation using anti-EGFR antibody (Upstate, Lake Placid, NY) for 16 h at 4C. After extensive washing, proteins were eluted from the immunoprecipitation pellet using 1x SDS buffer. The eluted samples were separated by electrophoresis on 7.5 % polyacrylamide gel and transferred to PVDF membrane. For immunoblotting, anti-phosphotyrosine monoclonal antibody (PY20) (BD Transduction Lab., Palo Alto, CA) and anti-EGFR antibody were used. The migration of the EGF-R from A431 cells was used as a control.

The following materials and methods were employed in Examples 5 to 10.

Animals Rats of our Wistar-derived colony were provided with water and rat chow ad libitum and housed in air-conditioned rooms illuminated 14 h/day. The experiments were carried out in accordance with the principles and guidelines for the use of laboratory animals and approved by the Weizmann Institute of Sciences research animal committee.

Immature female wistar rats (23-24 days) were injected subcutaneously with 12 IU pregnant mare serum gonadotropins (PMSG) (Sanofi SNA, Libourne, France) to enhance preovulatory folliclular development. For explanting follicle-enclosed oocytes (FEOs) for culture, the animals were sacrificed 48-50 h after PMSG by using C02 and subsequent cervical dislocation.

Intrabursal injections and the Ovulatorv Response In-vivo Two days after PMSG treatment, the bursa of rats was injected with the indicated doses of AG1478 (Calbiochem, San Diego, CA, USA) or vehicle (Tsafriri et al.,

Endocrinology 124: 415-21 (1989) ) between 1230-1330h. For intrabursal injection, animals were anesthetized by a cocktail of ketamine (40-60 mg/kg) and diazepam (2-3 mg/kg), and one of the ovaries was exteriorized via a small lumbosacral incision. A 29-G needle was threaded into the ovarian bursa via adjoining fat pad.

The location of the injection was confirmed by observation of the swelling of the bursa. After injection of the inhibitor or vehicle (50 ul/bursa), the ovary was replaced into the abdominal cavity and the skin was sutured. The contralateral ovary served as control. After 30 min animals were treated with 5 IU hCG. Vehicle treated- animals were used as an additional control. Ovulated COCs collected 18 h after hCG treatment were isolated from the oviduct and counted under a dissecting microscope.

Culture of preovulatory follicles.

The preovulatory follicles were isolated 48 hours post PMSG injection as previously described (Tsafriri et al., J. Reprod. Fertil. 31: 39-50 (1972)). Follicle- enclosed oocytes (FEOs) (10-15/dish) were cultured in Leibovitz's L-15 medium (Gibco, Grand Island, NY, USA) supplemented with penicillin (100 U/ml), streptomycin (100 ug/ml) (Gibco) and 5% fetal calf serum (Sera-Lab, Crawley Down, England). FEOs were cultured at 37°C in a controlled atmosphere of 50% 02, 1.3% C02 and 48.7% N2. After 60 min pre equilibration, Ovine LH (10-100 ng/ml) (generously provided by Dr. A. F. Parlow and the National Hormone and Pituitary distribution Program, NIDDK, NIH, USA), AG1478 (10 uM) and AG43 (10 , uM) (Calbiochem) AR (10-1000nM) (R&D Systems, Minneapolis, USA), ER (1- 250nM) (R&D Systems) or BTC (10-100nM) (R&D Systems) was added. Galardin (GM 6001) (20 uM) (Biomol, Plymouth Meeting, PA, USA) or (2R)-2- [ (4- biphenylylsulfonyl) amino]-3-phenylpropionic acid (100-500 uM) (Calbiochem) was added to the culture 30 min prior to LH or ER stimulation. The same vehicle solution was included in control culture media. At the end of the culture period, the follicles were punctured to release and collect the cumulus oocyte complex under dissecting microscope. Oocyte maturation was assessed by scoring groups of 10-15 released oocytes using Nomarski interference microscopy. The mean SEM of the groups of oocytes for each treatment are given.

Semiquantative RT-PCR Analysis The expression of rat hyaluronan synthase-2 (rHAS-2), cycloxygenase-2 (rCOX-2), TNFa-stimulated gene 6 (rTSG-6), rAR, rER and rBTC was examined by relative semiquantiative RT-PCR. Total RNA was extracted from ovaries and pre- ovulatory follicles at the indicated time intervals, using Tri-reagent (Sigma, St. Louis, MO, USA). For each sample, 250 ng of RNA from ovaries or 60-80 ng of RNA from preovulatory follicles were reverse-transcribed using oligo dT primer (Promega, Madison, WA, USA) followed by PCR amplification. Specific primers were used in order to amplify the following cDNAs: HAS-2 (forward 5'-gcttgaccctgcctcatctgtgg-3' <BR> <BR> (SEQ ID NO. : 03), reverse 5'-ctggttcagccatctcagatatt-3' (SEQ ID NO. : 04) ), COX-2 (forward 5'-ctgcttttcaaccagcagttcc-3' (SEQ ID NO. : 12), reverse 5'- tctgcagccatttctttctctc-3' (SEQ ID NO. : 13) ), TSG-6 (forward 5'- cgaagcgaatctttaaatcccc-3' (SEQ ID NO. : 14), reverse 5'-ctaaaccgtccagctaagaac-3' <BR> <BR> (SEQ ID NO. : 15) ), AR (forward 5'-ccacaggggactatgactac-3' (SEQ ID NO. : 16),<BR> reverse 5'-ttacggcggagacaaagac-3' (SEQ ID NO. : 17) ), ER (forward 5'- ccaccttctacaagcagtatc-3' (SEQ ID NO. : 18), reverse 5'-tcactctctcgtattcttccc-3' (SEQ ID NO. : 19) ), BTC (forward 5'-ggtcttgtgattctccagtg-3' (SEQ ID NO. : 20), reverse 5'-<BR> cttccttcttctttttgcgatg-3' (SEQ ID NO. : 21) ) which amplified a 403,376, 394,408, 407 and 400 bp product respectively. A fragment of S-16 cDNA, which served as an internal standard, was amplified in parallel using the following primers : forward 5'- cgttcaccttgatgagcccatt-3' (SEQ ID NO. : 22), reverse 5'-tccaagggtccgctgcagtc-3' (SEQ ID NO. : 23) which amplified a 100 bp product.

Each band was scanned and quantified by Fluor-STM Multimager (Bio-Rad, Hercules, CA). Briefly, the same size rectangle was used to surround each band and its intensity was determined using the Quantity one version 4.2. 1 software adjusted for ethidium bromide gel. Background in blank region of the gel was subtracted from the total area of the screened samples.

Immunoblottina Samples (75 ug whole cell lysates per each lane) were separated by SDS 7.5% polyacrylamide gel electrophoresis and electophoretically transferred to a nitrocellulose membrane. The samples were immunoblotted with polyclonal anti- EGFR (Cell Signaling Technology, Inc., Beverly, MA) or polyclonal anti PY1068 EGFR (Cell Signaling Technology) as indicated in the legends. The polyclonal

antibodies for immunoblots were detected with horseradish peroxidase conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) antibodies. Immunoblotting was performed according to manufactures instructions.

Specific signals were visualized on Xray by enhanced chemiluminescence detection system (Pierce, Rockford, IL) and scanned and quantified by Fluor-STM Multimager (Bio-Rad, Hercules, CA) adjusted for X-ray film as described above.

Histology Ovaries after superovulation were excised and fixed in Bouin's solution over night, then washed with 70% ethanol and embedded in paraffin wax. Sections 5 um thick were mounted on Super-frost/PLUS Micro slides (Fisher Scientific, Pittsburgh, PA, USA), then rehydrated and stained with HE for 10 min and then washed.

Statistical Analysis Statistical analysis by'ANOVA, student's t-test and Chi-square test was performed whenever appropriate. Data were expressed as mean SEM of pooled results obtained from at least three replicate cultures and two separate experiments.

Otherwise the results of two replicates were given. For the analysis of oocyte maturation in vivo (Fig. 12) standard errors were calculated according to the method described (Stone et al., Biol. Reprod. 19: 585-92 (1978)). Values of P<0.05 were considered to be significant.

Example 1 DNA Hybridization The expression of the EGF network in the ovary during the periovulatory period in wild type mice and in a mouse model with defective ovulation was investigated using chip hybridization technology, (Conti et al., J. Biol. Chem. 278, 5493 (2003)). It was determined that mice lacking phosphodiesterase PDE4D are subfertile and LH signaling was disrupted (Park et. al., Mol. Endocrinol. 17,1117 (2003) ). It was further determined that most of the follicles in PDE4D-/-ovaries undergo luteinization in response to LH but do not ovulate, nor do they express genes involved in cumulus expansion/follicle rupture, including cyclooxigenase 2 (Park 2003). FIG. 1A demonstrates that in both mouse strains, HB-EGF mRNA was predominantly expressed in the ovary 48 h after PMSG priming. In addition, FIG. 1A shows that an ovulatory dose of hCG/LH brought about the rapid and transient

expression of AR, EPI, and BTC mRNAs within 1-3 h after the injection, whereas the expression of HB-EGF was downregulated. In contrast, the expression of mRNAs for these growth factors was markedly decreased in PDE4D-/-ovaries (FIG. 1A).

Example 2 Northern Blot Analysis with RNA Derived From Ovaries after hCG Stimulation The rapid and transient induction of mRNAs coding for these growth factors in granulosa cells was confirmed by Northern blot analysis with RNA derived from ovaries after hCG stimulation in vivo and is demonstrated in FIG. 1 B. AR mRNA is the first to appear upon LH/hCG stimulation, followed by EPI and BTC. Whereas AR and BTC expression is transient, EPI mRNA remains elevated up to 12 h after the gonadotropin injection. The expression of these genes is initially restricted to mural granulosa cells of preovulatory follicles and, with the exception of BTC, specific signal is not detectable in cumulus cells surrounding the oocyte, as shown in FIG.

1 C. Furthermore, FIG. 5 demonstrates the finding that a major reduction in expression of these mRNAs was observed in PDE4D-/-ovaries using in situ hybridization.

Example 3 Fluorescence-Activated Cell Sorting to Confirm Protein Expression Fluorescence-activated cell sorting was subsequently employed to confirm that the mRNAs for AR, EPI, and BTC is translated into proteins expressed on the surface of granulosa cells. Using an EPI-specific antibody and IgG as a control, FIG.

2 shows that little specific signal was observed in granulosa cells prior to the gonadotropin stimulation. Conversely, up to 30% of the granulosa cells became positive 3-4 h after hCG injection. Taken together with the reported detection of EGF-like activity in follicular fluid (Hsu et. al., Biochem. Biophys. Res. Commun.

147,242 (1987) ), the above data demonstrate that LH stimulation of preovulatory follicles induces the expression of EGF-related growth factors.

Example 4 Follicle Culture Model In view of the above findings, whether these growth factors reproduce some of the LH effects was then investigated by employing a follicle culture model that recapitulates most of the LH effects in vivo, including oocyte meiotic maturation and cumulus expansion (See Tsafrirri et. al., J. Reprod. Fert. 31,39 (1972) ) FIG. 3A demonstrates that in the follicle culture, LH stimulation promotes resumption of oocyte meiosis measured as germinal vesicle breakdown (GVBD). Although BTC was only partially effective, exposure of the follicles to AR and EPI induced meiotic maturation to the same extent as LH (FIG. 3A), albeit with an important difference (FIG. 3B). Whereas LH-induced GVBD was completed only after 4 h, EPI and AR induction was maximal in 2-3 h, as shown in FIG. 3B. This finding is consistent with the hypothesis that AR and EPI function distal to LH, and the 1.5 h delay is attributed to the time required for LH to induce expression of the growth factors. It was further discovered that BTC mRNA is expressed with a time course incompatible with induction of oocyte maturation, because'it accumulates only 3 h after hCG stimulation, when oocyte maturation is well underway both in vivo and in vitro.

It is well known that LH/hCG stimulation of intact follicles causes cumulus expansion within 10 h of stimulation (see Sa'lustrai et. al., Zygote 4,313 (1996); Epigg, J. Exp. Zool. 208,345 (1979) ), a phenomenon reproduced by each of the three EGF-related growth factors, AR, EPI, and BTC, yet with a faster time course, as shown in FIG. 3C. This remodeling of the extracellular matrix is known to be preceded by the expression of Cox2, Has2, and Tsg6 genes shown to be essential for this process. FIG. 3D demonstrates that the mRNAs for these proteins were all induced by the EGF-related growth factors. In a simplified model of cumulus-oocyte complexes (COCs) where mural granulosa cells are removed, LH no longer affects cumulus expansion, but these EGF-related growth factors were even more effective than in intact follicles. COC isolated from preovulatory follicles display a compact structure with cells tightly packed around the oocyte (Fig. 3C). AR, EPI, and BTC all produce marked cumulus expansion at concentrations of 0.1-1 nM (FIG. 3C). Thus, these growth factors are among the most potent stimuli for cumulus expansion.

It has been reported that in vitro EGF stimulates cumulus expansion (see Trafriri (1972); Tirone et. al., J. Biol. Chem. 272,2787 (1997); Dekel et. al., Endocrinology 116,406 (1985) ). However, the physiological significance of this stimulation was not appreciated until now because the endogenous ligands for the ErbB receptors were unknown. As demonstrated in FIG. 6, inhibition of EGFR tyrosine kinase with the specific inhibitor AG1478 completely blocked the effects of the growth factors, confirming that their action is mediated by EGFR.

It was determined that given the induction of AR, EPI, and BTC in the follicle, LH/hCG should produce a delayed activation of the EGFR. Indeed, in vivo treatment with hCG caused EGFR phosphorylation that reached a maximum 3-4 h after injection, as shown in FIG. 4A. It was also found that in follicle cultures, the specific inhibitors AG1478 and compound 56 (C56) completely blocked the LH effects on cumulus expansion and oocyte maturation with EC50 of 300-500 nM, concentrations identical to those required to block EPI-induced maturation (FIG. 6). These inhibitor compounds are not toxic for the oocyte because spontaneous maturation, which occurs when the oocyte is removed from the follicle (COC), is not affected, as shown in FIG. 4B. Moreover, as shown in FIG. 4D, the inhibitors did not prevent the induction of AR mRNA, suggesting that the initial LH signaling is not affected.

Taken together, these experiments demonstrate that EGF-related growth factors induced by LH are sufficient to switch on cumulus expansion and oocyte maturation, and their signaling through EGFR is necessary for LH action. On the basis of these findings, the EGF-related growth factors are paracrine mediators of the LH signals during ovulation. This finding explains prior observations that EGF mimics some of the LH in vitro effects. Therefore, the above results indicate that the EGF-related growth factors, AR, EPI, and BTC, are the endogenous ligands that activate the EGFR in the follicle during the LH surge in a wide range of species.

It was previously observed that in mouse strains with inactivation of AR and BTC genes are available ; however, no reproductive phenotype was observed (Luetteke et. al., Development 126,2739 (1999); Jackson et al., EMBO J. 22,2701 (2003) ). Given the extreme redundancy observed for the EGF network, these findings are not inconsistent with the discovery of the present invention. Our data show that AR, EPI, and BTC expression and their effects are overlapping, suggesting a redundant mechanism. Whereas inactivation of the EGFR produces a

profound disruption of embryonic development, only double or triple knockouts of EGF-related growth factors will provide significant phenotypes in vivo.

Example 5 Stimulation of Follicular EGF-Like Factor mRNA bv LH To test the involvement of EGF-like factors in the LH pathway, FEO cultures were initially treated with 100 ng/ml LH for 3 and 9 hours. At the end of culture, levels of rER, rAR and rBTC mRNA were determined by semi-quantative RT-PCR.

LH brought about transient expression of rAR, rER and rBTC mRNA with a maximum at 3 hr followed by a marked decrease at 9 hr of culture. While LH further stimulated the basal BTC expression, the results show that it induced both AR and ER that were undetected in unstimulated follicles (Fig. 7).

Example 6 Effect of EGF-Like Factors on Meiosis In view of the above findings, it was next examined whether the addition of EGF-like factors, such as ER, AR and BTC in vitro reproduces some actions of LH.

Complete (with AR and ER), or partial (BTC) stimulation of the resumption of meiosis in FEO was obtained (Fig. 8, Panel A). ER was more potent in causing maximal maturation (88% GVB) at the concentration of 100 nM. Ten-fold higher dose of AR was required to reach a similar maturation rate.

Example 7 EGF Signal Transduction and LH Action To examine the role of EGF-signal transduction in LH stimulation of meiosis in vitro, the effect of the EGFR kinase inhibitor, AG1478, on LH-stimulated follicles was then tested. For this purpose, follicles were cultured with LH and AG1478.

AG1478, but not AG43 its inactive analog, blocked the LH-induced EGFR phosphorylation (Fig. 9, Panels A and B) and oocyte maturation (Fig. 8, Panel B, and Fig. 3, Panel C) (1% GVB with AG1478 vs. >88% with LH or LH+ AG 43) as compared to control follicles (4% GVB).

In order to examine the inhibitory effects of AG1478 in vivo, it was injected into the ovarian bursa before hCG administration (Fig. 10). Local administration of

3 ug/bursa into the periovarian sac resulted in 51% (14.5 +/-2.6 ova/ovary) inhibition of ovulation in treated ovary, as compared to untreated contralateral ovary (28.5 +/-2.3 ova/ovary) (p<0.0005), or to vehicle treated ovaries, (21.3 +/-2. 3 ova/ovary) (p<0. 02). Intra bursal administration of lower or higher concentrations of AG1478 revealed no significant differences in ovulation rate between treated ovaries and untreated ones or vehicle treated ovaries.

The results show that injection of AG1478 into the ovarian bursa provides for mediation of LH-stimulated ovulation by paracrine EGF-ligands. Thus the drug significantly inhibited the ovulation from the treated ovaries, as compared to untreated contra-lateral ones or vehicle-treated ovaries. This effect was biphasic, a dose lower than 3 ug per bursa was ineffective, as were higher doses.

Example 8 Histological appearance The histological appearance of the AG1478-treated ovaries with the effective dose of 3pg per bursa, was examined and compared to the untreated contralateral ovaries (Fig. 11). Most of the large antral follicles of control ovaries underwent follicular rupture during the ovulation processes and the mature ovum was released (Fig. 11, Panel A). Confirming the counts of ovulated oocytes, the histological examination of AG1478-treated ovaries revealed oocytes entrapped in the majority of the large antral follicles (Fig. 11, Panel B). These included GV oocytes with immature unexpanded cumuli (Fig. 11, Panel C), GVB oocytes at the first metaphase/anaphase (Fig. 11, Panel D) and oocytes that completed the first meiotic division as evidenced by the presence of the polar body (Fig. 11, Panel E). In control ovaries entrapped oocytes were also found in large antral follicles at different morphological stages (Fig. 12). Therefore, the number of large antral follicles with entrapped oocytes were analyzed quantitatively, as well as their meiotic status. The serial ovarian sections were scored for oocytes entrapped and for the ratio of immature oocytes in large antral follicles. The control and AG1478 treated ovaries contained 35% and 62% large follicles with entrapped oocytes, respectively (Chi- test p<0.0005) (Fig. 12). Moreover, the ratio of entrapped immature oocytes (GV) in the inhibitor treated ovaries was 5-fold higher as compared to the contralateral untreated ovaries (Chi-test p<0.0005).

The results show that in the ovaries treated with the effective dose of the inhibitor the large preovulatory follicles with entrapped oocytes had a markedly higher proportion of immature, GV oocytes, as compared to the oocytes entrapped in control ovaries. In other large follicles with entrapped oocytes, the meiotic process has been resumed and in some the first meiotic division was even completed as evidenced by the first polar body. Such separation between ovarian responses has been already observed in response to gonadotropic stimulation, resumption of meiosis having a lower threshold than completion of the first meiotic division and follicle rupture requiring even a stronger stimulus (Tsafriri et al., Prostaglandins 3: 461-7 (1973); Dekel et al., Fertil Steril 64: 1023-8 (1995) ). Thus, the in vivo studies show the physiological mediation of LH action on ovulation by ovarian EGF-like ligands.

Cumulus maturation or expansion involves the formation of a mucoid extracellular matrix by cumulus cells. The matrix is comprised mostly of the glycosaminoglycan hyaluronan (HA) forming a structural backbone as well as several hyaluronan binding proteins: the pròteoglycan versican, the serum-derived factor inter-a-trypsin inhibitor (lal) and the secreted protein TSG-6 (Mukhopadhyay et al., Arch Biochem Biophys 394: 173-81 (2001)). Cumulus expansion and HA synthesis are required for the release of fertilizable ova at ovulation (Chen et al., Mol Reprod Dev 34: 87-93 (1993) ). Likewise TSG-6 is also essential for ovulation since TSG-6 null female mice have a markedly lower ovulation rate than normal (Fulop et al., Development 130: 2253-61 (2003)).

Example 9 Expression of rHAS-2, rCOX-2 and rTSG-6 mRNA in follicles in culture Several genes, like COX-2, HAS-2 and TSG-6, have been previously shown to be essential for the ovulatory response, including cumulus expansion. Therefore, mRNA expression after LH and ER stimulation of follicles in culture was tested. It was found that both LH and ER induced the expression of rCOX-2, rHAS-2 and rTSG-6 mRNA (Fig. 13, upper panel), but ER was less effective than LH. To test whether LH action on some of the key genes associated with ovulation is mediated by the EGF-signal transduction network, the effect of AG1478 was then tested.

Therefore, the expression of rHAS-2, rCOX-2 and rTSG-6 mRNA in FEO treated by

LH or LH plus AG1478 after 6 hr incubation was compared. Very low expression of the tested enzymes was detected in control group ; however, LH stimulated mRNA expression of rHAS-2, rCOX-2 and TSG-6 with 3.8, 27 and 7. 3-fold increase, respectively. By contrast, AG1478 suppressed the LH-stimulated expression of these enzymes (Fig. 14). The expression of these enzymes was tested also in ovaries treated by AG1478 administration into the periovarian sac. The relative semi-quantitative RT-PCR revealed no significant differences in the ovarian expression of rHAS-2, rCOX-2 and rTSG-6 mRNAs between AG1478 treated and untreated-contralateral ovary in vivo (Fig. 15). Nevertheless, the expression of respective mRNAs was lower when compared to vehicle treated control levels (p<0.025, p<0.025 and p<0.05 respectively).

The results show that the LH surge triggers a cascade of inflammation- related responses, including the induction of COX-2 and the synthesis of prostaglandins. Pharmacological inhibition of COX-2 in several mammalian species suppresses ovulation (Tsafriri et al., Exp Clin Endocrinol Diabetes 107: 1-11 (1999)) and mice with null-mutation for either COX-2 or the prostaglandih E2 (PGE2) receptor subtype EP2 have impaired ovulation associated with defective COC expansion (Davis et al., Endocrinology 140: 2685-95 (1999); Richards et al., Recent Prog Horm Res 57: 195-220 (2002) ). Expression of COX-2 and the resulting prostaglandins is involved at two steps required for the ovulation of a fertilizable ovum: cumulus expansion (Joyce et al., Endocrinology 142: 3187-97 (2001) ) and follicle rupture, most probably through activation of collagenolytic enzymes (Tsafriri et al., Exp Clin Endocrinol Diabetes 107: 1-11 (1999) ). These two responses could be separated by the inhibition of hyaluronic acid synthesis, which prevented cumulus expansion but not rupture of the follicle wall and the mature oocytes remained entrapped within the ruptured follicles (Chen et al., Mol Reprod Dev 34: 87-93 (1993) ). The precise interactions of prostaglandins, cumulus expansion and follicle rupture remain to be elucidated. The examination of the follicular expression of the three genes HAS-2, TSG-6 and COX-2 essential for these ovulatory changes provided molecular markers for stimulation of ovulation by LH/hCG and the role of paracrine EGF family members in mediating this response.

This is especially valuable in cultured rat follicles that do not undergo rupture.

LH/hCG and ER stimulated follicular mRNA of HAS-2, TSG-6 and COX-2 in vitro.

Further, AG1478 inhibited the expression of these mRNA species in response to LH/hCG in cultured follicles and also in vivo in the whole ovary. This latter result is of special interest. The results (as shown in Fig. 15) show that both ovaries, the one injected with the inhibitor and the untreated contralateral ovary showed similar reduction in the expression of these three marker RNAs when compared to the control animals injected with the vehicle only. Yet, there was a significant reduction in ovulation rate of the AG1478-treated ovaries compared to the untreated ovaries or vehicle treated controls. These results show that the drug reached the untreated ovary as well.

Example 10 Effects of Galardin on the LH and ER-induced maturation of follicle-enclosed oocytes To test whether the action of LH on resumption of meiosis is dependent upon proteolytic activity, suggested to be required for the release of cell-membrane bound EGF-like factors (Roelle et al., J Biol Chem 278: 47307-18 (2003) ), a broad spectrum metalloprotease inhibitor was used, Galardin and a potent MMP-2/MMP-9 inhibitor ( (2R)-2- [ (4biphenylsulfonyl) amino]-3-phenylpropionic acid). Only Galardin (20, uM) prevented the LH-induced resumption of meiosis in FEO in culture, but not that of exogenous ER (Fig. 16). By contrast, the specific MMP-2/MMP-9 inhibitor (100-500 uM) did not affect resumption of meiosis (>80% GVB).

The results show that the broad-spectrum metalloprotease inhibitor Galardin, but not a specific MMP-2/MMP-9 inhibitor, prevented the stimulation of the resumption of meiosis in rat follicles by LH. Thus the reulsts demonstrate in a physiological unit comprised of several cell types the utilization of TMPS mechanisms and trans-activation of RTK by the stimulation of GPCR by LH. The failure of a specific MMP-2/MMP-9 inhibitor to block this activity of LH is of interest, since they were implicated in the trans-activation of the EGFR by GnRH in mouse gonadotropes cell line (Roelle et al., J Biol Chem 278: 47307-18 (2003)).

It is evident from the above results and discussion that the subject invention provides an important new way of modulating ovulation in a subject. As such, the subject invention provides important new ways of treating infertility. The subject

invention also provides important new ways of achieving contraception. Accordingly, the subject invention represents a significant contribution to the art.

All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

The preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i. e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims.