COMPOSITIONS AND METHODS FOR DOLLAR I(IN VITRO) FERTILIZATION

Abstract: Compositions and methods are described to enhance embryo implantation. Cytokines capable of binding to a receptor complex (the complex comprising a member of the hematopoietic cytokine receptor family) are utilized to treat female mammals, and in particular, females receiving in vitro fertilized embryos.

Description: COMPOSITIONS AND METHODS FOR IN VITRO FERTILIZATION FIELD OF THE INVENTION The present invention relates to compositions and methods for in vitro fertilization (IVF). and in particular, enhancing implantation frequencies and blocking implantation in mammals (including but not limited to humans) using LIF and LIF antagonists, respectively. BACKGROUND Infertility is a problem that afflicts approximately 9% of couples in the United States who wish to have children. The causes of infertility are many and varied. Whatever the cause in the particular case, treatment can be very expensive. Indeed, it is estimated that the annual cost of treating infertile couples in the United States is approximately 1 billion dollars. Frequently, treatment involves the use of in vitro fertilization (IVF) in which eggs from the woman are recovered and fertilized with the male's sperm. The resulting embryo(s) are then surgically transferred back to the woman's uterus for development to continue. However, only about 25% (at best) of the transplanted embryos implant properly with even fewer resulting in live birth. Failure of transplanted embryos to implant is thus a major difficulty in treating the larger problem of infertility. One obvious approach to improving the chance of success is to increase the number of embryos being implanted. While the use of multiple embryos {e.g. the transfer of up to four embryos to the uterus at the same time) increases the chances of one of them implanting, the recipient also runs the risk of multiple implants and consequently multiple births. See Eisner et al., Am J Obstet Gynecol 177:350-355 (1997). This method is also unsatisfactory in that if the uterus is physically and chemically unprepared to receive embryos because the hormonal regulation is dysfunctional, simply transplanting more embryos will not solve the problem. A number of hormones {e.g. estrogen, progesterone, etc.) have been identified as having a role in the preparation of the uterus to receive a transplanted embryo, and thus many attempts to improve implantation frequency have focused on the regulation of these hormones. For example, estrogen appears to play a significant role in regulating uterine receptivity to embryo implantation, and thus one treatment of implantation failure is to regulate (e.g. increase) the estrogen level of the host. This approach to treatment frequently backfires, however, as it is thought that this can cause implantation failure due to improper uterine physiology and chemical receptivity induced by the estrogen treatment. Moreover, increased levels of estrogens employed during IVF treatment have been implicated in severe complications of the Ovarian Hyperstimulation Syndrome (OHS). See e.g. MacDougall et ai. Hum Reprod 7:597-600 (1992). What is needed is an effective substitute for the standard hormonal treatment currently used to increase uterine receptivity to embryo implantation. A one-time, non- invasive direct administration of an estrogen substitute would allow increased use and effectiveness of treatment of implantation failure, without the side effects of increased estrogen levels. DEFINITIONS The term "hematopoietic cytokine receptor family" is the recognized family of receptors \see e.g. T. Taga and T. Kishimoto, "Cytokine receptors and signal transduction." FASAB J. 6:3387 ( 1992)] having structural similarity to IL-6-R, and more particularly, having structural similarity with respect to the approximately 200 amino acid-homologous regions of IL-6-R. said region characterized by four conserved cysteine residues in the amino-terminal half and a WSXWS motif at is carboxyl-terminal end (wherein W is Trp; S is Sen and X is a nonconserved amino acid). Members of this family include IL-2-R. 1L-3-R. IL-4-R. IL-5-R. IL-6-R. IL-7-R. IL-9-R, EPO-R. G-CSF-R. GM-CSF-R and LIF- R. A subset of the above-named receptors typically exist as a "receptor complex" comprising the above-named molecule in association with another protein called gpl 30. Thus, the corresponding ligands that bind the above-named receptors typically bind the complex. Thus, the present invention contemplates "a cytokine capable of binding to a receptor complex comprising a member of the hematopoietic cytokine receptor family." Examples of such ligands are IL-6 and LIF. Other examples include Oncostatin M, Ciliary Neurotrophic Factpr and Cardiotrophin-1. The present invention also contemplates molecules that are simply capable of binding to gp l30, such molecules useful for the treatment of females to enhance implantation frequencies. An "estrogen-free" composition contains no detectable estrogen (e.g. detectable by immunological assay). A "substantially estrogen-free" composition contains only background amounts of estrogen. A female "subsequently" not treated with estrogen is a female who. subsequent to the claimed method step (e.g. the step of introducing an embryo into said female), does not receive estrogen. A female "that has not been treated with estrogen" is a female who has received no exogenous estrogen prior to the first step of the claimed method. SUMMARY OF THE INVENTION The present invention relates to compositions and methods for in vitro fertilization (IVF). and in particular, enhancing implantation frequencies and blocking implantation in mammals (including but not limited to humans) using LIF and LIF antagonists, respectively. With respect to enhancing implantation frequencies, the present invention contemplates treatment of females with the cytokine Leukemia Inhibitory Factor (LIF). The present invention contemplates the direct injection of LIF. which leads to the embryo ' s normal development to birth and also, the use of LIF as a replacement to using estrogen for in vitro fertilization. It is not intended that the present invention be limited to only one type of LIF. A variety of LIF proteins are contemplated, including but not limited to LIF from human (SEQ ID NO: l ), murine (SEQ ID NO:2), rat (SEQ ID NO:3), porcine (SEQ ID NO:4) and ovine (SEQ ID NO:5) (see Figure 1). In a preferred embodiment, the LIF administered according to the present invention is recombinantly produced LIF. It is also not intended that the present invention be limited to LIF. In one embodiment, the present invention contemplates a method, comprising: a) providing i) a female to be implanted with at least one embryo, and ii) an estrogen-free composition, comprising a cytokine capable of binding to a receptor complex comprising a member of the hematopoietic cytokine receptor family; and b) administering said estrogen-free composition to said female under conditions such that said embryo implants. In a preferred embodiment, said cytokine is LIF and said embryo is an in vitro fertilized embryo. In a preferred embodiment, the present invention contemplates a method, comprising: a) providing i) an embryo, ii) a female to be implanted with said embryo, and iii) an estrogen-free composition, comprising a cytokine capable of binding to a receptor complex comprising a member of the hematopoietic cytokine receptor family; b) introducing said embryo into said female, said female subsequently not treated with exogenous estrogen (i.e. estrogen given exogneously) and c) administering said estrogen- free composition to said female under conditions such that said embryo implants. In a preferred embodiment, said cytokine is LIF and said embryo is an in vitro fertilized embryo. It is also not intended that the present invention be limited to LIF. In one embodiment, the present invention contemplates a method, comprising: a) providing i) a female that has not been treated with exogneous estrogen, said female to be implanted with at least one embryo, and ii) an estrogen-free composition, comprising a cytokine capable of binding to a receptor complex comprising a member of the hematopoietic cytokine receptor family; and b) administering said estrogen-free composition to said female under conditions such that said embryo implants. In a preferred embodiment, said cytokine is LIF and said embryo is an in vitro fertilized embryo. In another embodiment, the present invention contemplates a method, comprising: a) providing i) a female that has not been treated with exogenous estrogen, said female to be implanted with at least one embryo, and ii) an estrogen-free composition, comprising a ligand capable of binding to gpl30: and b) administering said estrogen-free composition to said female under conditions such that said embryo implants. In a preferred embodiment. said embryo is an in vitro fertilized embryo. DESCRIPTION OF THE DRAWINGS Figure 1 shows an alignment of the prediced amino acid sequence of human (SEQ ID NO: l ). murine (SEQ ID NO:2), rat (SEQ ID NO:3), porcine (SEQ ID NO:4) and ovine (SEQ ID NO:5) versions of LIF (the predicted signal sequence is shown by a solid line above the sequence and potential N-linked glycosylation sites are indicated by #). Figure 2 shows the nucleic acid sequence (SEQ ID NO:6) and amino acid sequence (SEQ ID NO:7) of gpl30. Figure 3 schematically shows the cDNA encoding a fusion protein of IL-6 and sIL- 6R. DESCRIPTION OF THE INVENTION The present invention relates to compositions and methods for in vitro fertilization (IVF). and in particular, enhancing implantation frequencies and blocking implantation in mammals (including but not limited to humans) using LIF and LIF antagonists. respectively. With respect to enhancing implantation frequencies, the present invention contemplates treatment of females with the cytokine Leukemia Inhibitory Factor (LIF). LIF has been identified as having a role in the implantation process and attempts have been made to increase implantation frequency by either culturing the embryos in LIF-containing medium (see e.g. U.S. Patent No. 5.366,888 to Fry et al.. hereby incorporated by reference) or continuously administering LIF to the host. See Stewart et ai . Nature 359:76-79 (1992). These techniques have met with some success, but are inferior in that surgery is involved in the implantation of a micro-pump and that they do not address the problem caused by the estrogen treatment. A. Leukemia Inhibitory Factor (LIF) And The LIF Receptor LIF is a member of a family of proteins (the IL-6 family of cytokines) that show some structural similarities. They sometimes have similar effects on the same cell, because they can bind to each other's receptors and stimulate them. What all these factors have in common is that for their specific receptors to function properly, it appears that they have to associate with the gpl30 protein. Without being limited to any particular mechanism, the present invention contemplates that other members of the IL-6 family of cytokines will have the same effects on embryo implantation as LIF. and in particular, those members that require the LIF receptorα are expected to be particularly effective. The receptor complex that LIF binds to at the cell surface is comprised of two proteins: the LIF receptorα and another protein called gpl30 (see Figure 2). When LIF binds to the LIF receptorα this complex then binds to gpl30 which causes the receptor to "fire" so activating a number of processes and responses in the cells to which LIF has bound. While the present invention is not limited to any particular mechanism, this association of LIF + LIF receptorα with gpl 30 appears to be essential for the whole receptor complex to function properly. As noted above, the present invention contemplates the administration of ligands capable of binding gpl30. Such compounds can be readily screened for and tested in a binding assay with gpl30. B. LIF Antagonists In one embodiment, the present invention relates to compositions and methods for blocking implantation in mammals (including but not limited to humans) using LIF antagonists. A variety of LIF antagonists are contemplated, including but not limited to mutant versions of LIF and antibodies to LIF. Mutant LIF can be LIF with amino acid modifications, substitutions and deletions. In one embodiment. LIF is altered such that it cannot bind to the LIF receptor complex. Such altered LIF molecules are readily tested (usually by labeling them) for binding to the LIF receptor on cells. Such an altered LIF molecule can then be used to block implantation in normal female mammals (e.g. females having normal levels of functional LIF). C. Formulations And Preparations It is not intended that the present invention be limited by the particular nature of the therapeutic preparation used for administration of the cytokines useful for enhanced implantation. For example, such compositions can be provided together with physiologically tolerable liquid, gel or solid carriers, diluents, adjuvants and excipients. These therapeutic preparations can be administered to mammals for veterinary use, such as with domestic animals, and clinical use in humans in a manner similar to other therapeutic agents. In general, the dosage required for therapeutic efficacy will vary according to the type of use and mode of administration, as well as the particularized requirements of individual hosts. Such compositions are typically prepared as liquid solutions or suspensions, or in solid forms. Oral formulations for cancer usually will include such normally employed additives such as binders, fillers, carriers, preservatives, stabilizing agents, emulsifiers, buffers and excipients as. for example, pharmaceutical grades of mannitol. lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations, or powders, and typically contain l%-95% of active ingredient, preferably 2%-70%. The compositions are also prepared as injectables. either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in. liquid prior to injection may also be prepared. The antagonists of the present invention are often mixed with diluents or excipients which are physiological tolerable and compatible. Suitable diluents and excipients are, for example, water, saline, dextrose, glycerol. or the like, and combinations thereof. In addition, if desired the compositions may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, stabilizing or pH buffering agents. Additional formulations which are suitable for other modes of administration, such as topical administration, include salves, tinctures, creams, lotions, and, in some cases, suppositories. For salves and creams, traditional binders, carriers and excipients may include, for example, polyalkylene glycols or triglycerides. EXPERIMENTAL The following examples serve to illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof. EXAMPLE 1 In this example, LIF is shown to be useful as a replacement for estrogen. Mice were ovariectomized and allowed to recover for 18 days before starting experimental manipulation. Priming of the uterus by steroid hormones was started by the injection of 1 OOng of Estrogen on each of 3 consecutive days. On the 6th day the mice were given a single injection of Progesterone in the form of Depo-Provera. Three days later an average of 6 blastocvsts. either directly isolated from females or from cultures started the previous day when the embryos were at the 8-cell stage were surgically transferred to the right uterine horn. After the mice had recovered from surgery they were either given 1 injection of 40ng of estrogen or 3 injections, 1 hour apart, of 5μg LIF. Four days later (equivalent to Day 7-8 of pregnancy) the mice were sacrificed for analysis, and the implantation sites recovered. Examination of the deciduas revealed that they contained morphologically normal embryos at the right stage of development. The results presented in Table 1 using this particular experimental protocol reveal that the injection of LIF can unequivocally replace the injection of estrogen for inducing embryo implantation. In turn this demonstrates that LIF could be used as an alternative to already established hormonal treatments for increasing the frequency of obtaining successful implantations and viable embryonic development in the treatment of human infertility following In Vitro Fertilization and transfer of the resulting embryo to the mother ' s uterus. Table 1 Injection of Recombinant LIF Substitutes for Nidatory (implantational) Estrogen at inducing Blastocyst Implantation EXAMPLE 2 In this example, a soluble IL-6 ligand/receptor fusion protein (sIL-6RFP) is tested. The sequences for IL-6 and IL-6R are known. See e.g. K. Yamasaki et al.. "Cloning and expression of the human Interleukin-6 (BSF-2/IFN β2) receptor" Science 241 :825-828 (1988). A cassette (Figure 2) consisting of human sIL-6R cDNA (corresponding to amino acid residues 1 13-323) and human IL-6 cDNA (corresponding to amino acid residues 29- 212) fused by a synthetic DNA linker coding for the amino acid sequence Arg-Gly-Gly- Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Val-Glu was constructed by the PCR technique and cloned into the Pichia pastoris expression vector pPIC9 (Invitrogen. San Diego, CA) using the restriction enzymes SnaBI and EcoRl. Cleavage of the signal peptide in transfected yeast cells leads to the secretion of the fusion protein with an NH : -terminal extension of 8 amino acid residues (Glu-Lys-Arg-Glu-Ala-Glu-Ala-Tyr). The fusion protein was purified from yeast supernatants by anion-exchange chromatography and gelfiltration and visualized by SDS-PAGE and silver staining. This fusion protein is essentially a hybrid protein comprised of IL-6 already bound to its specific receptor. This complex will then bind to any cell that has gpl30 and activate the cell. To test the protein, pseudopregnant wild type mice were ovariectomized on Day 3 of pregnancy. Six females had ten 8-cell stage embryos surgically transferred to their right uterine horn and were injected with progesterone. Five days later 3 mice were each injected with 2μg of sIL-6RFP and the other 3 were injected with a saline control. Three days later the mice were examined for the presence of implanted embryos. The results are shown in Table 2. Table 2 The results show that this fusion protein is effective at inducing embryo implantation in the absence of estrogen, and thus can be used as an alternative to LIF. SEQUENCE LISTING <110> Stewart, Colin L. Shatzer, Teresa Sullivan, Teresa Chen, Jim-Ray Hernandez, Lid a <120> COMPOSITIONS AND METHODS FOR IN VITRO FERTILIZATION <130> NIH-05034 <140> not yet assigned <141> 1999-07-02 <150> 60/091,771 <151> 1998-07-06 <160> 9 <170> Patentln Ver. 2.0 ,210> 1 <211> 202 <212,> PRT '213> Homo sapiens <400> 1 Met Lys Val Leu Ala Ala Gly Val Val Pro Leu Leu Leu Val Leu His 1 5 10 15 Trp Lys His Gly Ala Gly Ser Pro Leu Pro lie Thr Pro Val Asn Ala 20 25 30 Thr Cys Ala lie Arg His Pro Cys His Asn Asn Leu Met Asn Gin lie 35 40 45 Arg Ser Gin Leu Ala Gin Leu Asn Gly Ser Ala Asn Ala Leu Phe lie 50 55 60 Leu Tyr Tyr Thr Ala Gin Gly Glu Pro Phe Pro Asn Asn Leu Asp Lys 65 70 75 80 Leu Cys Gly Pro Asn Val Thr Asp Phe Pro Pro Phe His Ala Asn Gly 85 90 95 Thr Glu Lys Ala Lys Leu Val Glu Leu Tyr Arg lie Val Val Tyr Leu 100 105 110 Gly Thr Ser Leu Gly Asn lie Thr Arg Asp Gin Lys lie Leu Asn Pro 115 120 125 Ser Ala Leu Ser Leu His Ser Lys Leu Asn Ala Thr Ala Asp lie Leu 130 135 140 Arg Gly Leu Leu Ser Asn Val Leu Cys Arg Leu Cys Ser Lys Tyr His 145 150 155 160 Val Gly His Val Asp Val Thr Tyr Gly Pro Asp Thr Ser Gly Lys Asp 165 170 175 Val Phe Gin Lys Lys Lys Leu Gly Cys Gin Leu Leu Gly Lys Tyr Lys 180 185 190 Gin lie lie Ala Val Leu Ala Gin Ala Phe 195 200 <210> 2 <211> 203 <212> PRT <213> Mus musculus <400> 2 Met Lys Val Leu Ala Ala Gly lie Val Pro Leu Leu Leu Leu Val Leu 1 5 10 15 His Trp Lys His Gly Ala Gly Ser Pro Leu Pro lie Thr Pro Val Asn 20 25 30 Ala Thr Cys Ala lie Arg His Pro Cys His Gly Asn Leu Met Asn Gin 35 40 45 lie Lys Asn Gin Leu Ala Gin Leu Asn Gly Ser Ala Asn Ala Leu Phe 50 55 60 lie Ser Tyr Tyr Thr Ala Gin Gly Glu Pro Phe Pro Asn Asn Val Glu 65 70 75 80 Lys Leu Cys Ala Pro Asn Met Thr Asp Phe Pro Ser Phe His Gly Asn 85 90 95 Gly Thr Glu Lys Thr Lys Leu Val Glu Leu Tyr Arg Met Val Ala Tyr 100 105 110 Leu Ser Ala Ser Leu Thr Asn lie Thr Arg Asp Gin Lys Val Leu Asn 115 120 125 Pro Thr Ala Val Ser Leu Gin Val Lys Leu Asn Ala Thr lie Asp Val 130 135 140 Met Arg Gly Leu Leu Ser Asn Val Leu Cys Arg Leu Cys Asn Lys Tyr 145 150 155 160 Arg Val Gly His Val Asp Val Pro Pro Val Pro Asp His Ser Asp Lys 165 170 175 Glu Ala Phe Gin Arg Lys Lys Leu Gly Cys Gin Leu Leu Gly Thr Tyr 180 185 190 Lys Gin Val lie Ser Val Val Val Gin Ala Phe 195 200 210> 3 <211> 202 <212> PRT <213> Rattus norvegicus <400> 3 Met Lys Val Leu Ala Ala Gly lie Val Pro Leu Leu Leu lie Leu His 1 5 10 15 Trp Lys His Gly Ala Gly Ser Pro Leu Pro lie Thr Pro Val Asn Ala 20 25 30 Thr Cys Ala lie Arg His Pro Cys His Gly Asn Leu Met Asn Gin lie 35 40 45 Lys Ser Gin Leu Ala Gin Leu Asn Gly Ser Ala Asn Ala Leu Phe lie 50 55 60 Ser Tyr Tyr Thr Ala Gin Gly Glu Pro Phe Pro Asn Asn Val Asp Lys 65 70 75 80 Leu Cys Ala Pro Asn Met Thr Asp Phe Pro Pro Phe His Ala Asn Gly 85 90 95 Thr Glu Lys Thr Lys Leu Val Glu Leu Tyr Arg Met Val Thr Tyr Leu 100 105 110 Gly Ala Ser Leu Thr Asn lie Thr Trp Asp Gin Lys Asn Leu Asn Pro 115 120 125 Thr Ala Val Ser Leu Gin lie Lys Leu Asn Ala Thr Thr Asp Val Met 130 135 140 Arg Gly Leu Leu Ser Ser Val Leu Cys Arg Leu Cys Asn Lys Tyr His 145 150 155 160 Val Gly His Val Asp Val Pro Cys Val Pro Asp Asn Ser Ser Lys Glu 165 170 175 Ala Phe Gin Arg Lys Lys Leu Gly Cys Gin Leu Leu Gly Thr Tyr Lys 180 185 190 Gin Val lie Ser Val Val Val Gin Ala Phe 195 200 <-210> 4 <211> 202 <212> PRT <213> Sus scrofa <-400> 4 Met Lys Val Leu Ala Ala Gly Val Val Pro Leu Leu Leu Val Leu His 1 * 5 10 15 Trp Lys His Gly Ala Gly Ser Pro Leu Ser lie Thr Pro Val Asn Ala 20 25 30 Thr Cys Ala Thr Arg His Pro Cys His Ser Asn Leu Met Asn Gin lie 35 40 45 Lys Asn Gin Leu Ala His Val Asn Ser Ser Ala Asn Ala Leu Phe lie 50 55 60 Leu Tyr Tyr Thr Ala Gin Gly Glu Pro Phe Pro Asn Asn Leu Asp Lys 65 70 75 80 Leu Cys Gly Pro Asn Val Thr Asn Phe Pro Pro Phe His Ala Asn Gly 85 90 95 Thr Glu Lys Ala Arg Leu Val Glu Leu Tyr Arg lie lie Ala Tyr Leu 100 105 110 Gly Ala Ser Leu Gly Asn lie Thr Arg Asp Gin Arg Ser Leu Asn Pro 115 " 120 125 Gly Ala Val Asn Leu His Ser Lys Leu Asn Ala Thr Ala Asp Ser Met 130 135 140 Arg Gly Leu Leu Ser Asn Val Leu Cys Arg Leu Cys Asn Lys Tyr His 145 150 155 160 Val Ala His Val Asp Val Ala Tyr Gly Pro Asp Thr Ser Gly Lys Asp 165 170 175 Val Phe Gin Lys Lys Lys Leu Gly Cys Gin Leu Leu Gly Lys Tyr Lys 180 185 190 Gin Val lie Ser Val Leu Ala Arg Ala Phe 195 200 <210> 5 <211> 202 <212> PRT <213> Ovis aries <^400> 5 Met Lys lie Leu Ala Ala Gly Val Val Pro Leu Leu Leu Val Leu His 1 5 10 15 Trp Lys Pro Gly Ala Gly Ser Pro Leu Pro lie Asn Pro Val Asn Ala 20 25 30 Thr Cys Asn Thr His His Pro Cys Pro Ser Asn Leu Met Ser Gin lie 35 40 45 Arg Ser Gin Leu Ala Gin Leu Asn Gly Thr Ala Asn Ala Leu Phe lie 50 55 60 Leu Tyr Tyr Thr Ala Gin Gly Glu Pro Phe Pro Asn Asn Leu Asp Lys 65 70 75 80 Leu Cys Gly Pro Asn Val Thr Asp Phe Pro Pro Phe Gin Pro Asn Gly 85 90 95 Thr Glu Lys Val Arg Leu Val Glu Leu Tyr Arg lie Val Ala Tyr Leu 100 105 110 Gly Thr Ala Leu Gly Asn lie Thr Arg Asp Gin Lys Thr Leu Asn Pro 115 120 125 Thr Ala His Ser Leu His Ser Lys Leu Asn Ala Thr Ala Asp Thr Leu 130 135 140 Arg Gly Leu Leu Ser Asn Val Leu Cys Arg Leu Cys Ser Lys Tyr His 145 150 155 160 Val Ala His Val Asp Val Ala Tyr Gly Pro Asp Thr Ser Gly Lys Asp 165 170 175 Val Phe Gin Lys Lys Lys Leu Gly Cys Gin Leu Leu Gly Lys Tyr Lys 180 185 190 Gin Val Met Ala Val Leu Ala Gin Ala Phe 195 200 <210> 6 <211> 3085 <212> DNA <213> Homo sapiens <400> 6 gagcagccaa aaggcccgcg gagtcgcgct gggccgcccc ggcgcagctg aaccgggggc 60 cgcgcctgcc aggccgacgg gtctggccca gcctggcgcc aaggggttcg tgcgctgtgg 120 agacgcggag ggtcgaggcg gcgcggcctg agtgaaaccc aatggaaaaa gcatgacatt 180 tagaagtaga agacttagct tcaaatccct actccttcac ttactaattt tgtgatttgg 240 aaatatccgc gcaagatgtt gacgttgcag acttgggtag tgcaagcctt gtttattttc 300 ctcaccactg aatctacagg tgaacttcta gatccatgtg gttatatcag tcctgaatct 360 ccagttgtac aacttcattc taatttcact gcagtttgtg tgctaaagga aaaatgtatg 420 gattattttc atgtaaatgc taattacatt gtctggaaaa caaaccattt tactattcct 480 aaggagcaat atactatcat aaacagaaca gcatccagtg tcacctttac agatatagct 540 tcattaaata ttcagctcac ttgcaacatt cttacattcg gacagcttga acagaatgtt 600 tatggaatca caataatttc aggcttgcct ccagaaaaac ctaaaaattt gagttgcatt 660 gtgaacgagg ggaagaaaat gaggtgtgag tgggatggtg gaagggaaac acacttggag 720 acaaacttca ctttaaaatc tgaatgggca acacacaagt ttgctgattg caaagcaaaa 780 cgtgacaccc ccacctcatg cactgttgat tattctactg tgtattttgt caacattgaa 840 gtctgggtag aagcagagaa tgcccttggg aaggttacat cagatcatat caattttgat 900 cctgtatata aagtgaagcc caatccgcca cataatttat cagtgatcaa ctcagaggaa 960 ctgtctagta tcttaaaatt gacatggacc aacccaagta ttaagagtgt tataatacta 1020 aaatataaca ttcaatatag gaccaaagat gcctcaactt ggagccagat tcctcctgaa 1080 gacacagcat ccacccgatc ttcattcact gtccaagacc ttaaaccttt tacagaatat 1140 gtgtttagga ttcgctgtat gaaggaagat ggtaagggat actggagtga ctggagtgaa 1200 gaagcaagtg ggatcaccta tgaagataga ccatctaaag caccaagttt ctggtataaa 1260 atagatccat cccatactca aggctacaga actgtacaac tcgtgtggaa gacattgcct 1320 ccttttgaag ccaatggaaa aatcttggat tatgaagtga ctctcacaag atggaaatca 1380 catttacaaa attacacagt taatgccaca aaactgacag taaatctcac aaatgatcgc 1440 tatctagcaa ccctaacagt aagaaatctt gttggcaaat cagatgcagc tgttttaact 1500 atccctgcct gtgactttca agctactcac cctgtaatgg atcttaaagc attccccaaa 1560 gataacatgc tttgggtgga atggactact ccaagggaat ctgtaaagaa atatatactt 1620 gagtggtgtg tgttatcaga taaagcaccc tgtatcacag actggcaaca agaagatggt 1680 accgtgcatc gcacctattt aagagggaac ttagcagaga gcaaatgcta tttgataaca 1740 gttactccag tatatgctga tggaccagga agccctgaat ccataaaggc ataccttaaa 1800 caagctccac cttccaaagg acctactgtt cggacaaaaa aagtagggaa aaacgaagct 1860 gtcttagagt gggaccaact tcctgttgat gttcagaatg gatttatcag aaattatact 1920 atattttata gaaccatcat tggaaatgaa actgctgtga atgtggattc ttcccacaca 1980 gaatatacat tgtcctcttt gactagtgac acattgtaca tggtacgaat ggcagcatac 2040 acagatgaag gtgggaagga tggtccagaa ttcactttta ctaccccaaa gtttgctcaa 2100 ggagaaattg aagccatagt cgtgcctgtt tgcttagcat tcctattgac aactcttctg 2160 ggagtgctgt tctgctttaa taagcgagac ctaattaaaa aacacatctg gcctaatgtt 2220 ccagatcctt caaagagtca tattgcccag tggtcacctc acactcctcc aaggcacaat 2280 tttaattcaa aagatcaaat gtattcagat ggcaatttca ctgatgtaag tgttgtggaa 2340 atagaagcaa atgacaaaaa gccttttcca gaagatctga aatcattgga cctgttcaaa 2400 aaggaaaaaa ttaatactga aggacacagc agtggtattg gggggtcttc atgcatgtca 2460 tcttctaggc caagcatttc tagcagtgat gaaaatgaat cttcacaaaa cacttcgagc 2520 actgtccagt attctaccgt ggtacacagt ggctacagac accaagttcc gtcagtccaa 2580 gtcttctcaa gatccgagtc tacccagccc ttgttagatt cagaggagcg gccagaagat 2640 ctacaattag tagatcatgt agatggcggt gatggtattt tgcccaggca acagtacttc 2700 aaacagaact gcagtcagca tgaatccagt ccagatattt cacattttga aaggtcaaag 2760 caagtttcat cagtcaatga ggaagatttt gttagactta aacagcagat ttcagatcat 2820 atttcacaat cctgtggatc tgggcaaatg aaaatgtttc aggaagtttc tgcagcagat 2880 gcttttggtc caggtactga gggacaagta gaaagatttg aaacagttgg catggaggct 2940 gcgactgatg aaggcatgcc taaaagttac ttaccacaga ctgtacggca aggcggctac 3000 atgcctcagt gaaggactag tagttcctgc tacaacttca gcagtaccta taaagtaaag 3060 ctaaaatgat tttatctgtg aattc 3085 <210> 7 <211> 918 <212> PRT ^213 > Homo sapiens <400> 7 Met Leu Thr Leu Gin Thr Trp Val Val Gin Ala Leu Phe lie Phe Leu 1 5 10 15 Thr Thr Glu Ser Thr Gly Glu Leu Leu Asp Pro Cys Gly Tyr He Ser 20 25 30 Pro Glu Ser Pro Val Val Gin Leu His Ser Asn Phe Thr Ala Val Cys 35 40 45 Val Leu Lys Glu Lys Cys Met Asp Tyr Phe His Val Asn Ala Asn Tyr 50 55 60 He Val Trp Lys Thr Asn His Phe Thr He Pro Lys Glu Gin Tyr Thr 65 70 75 80 He He Asn Arg Thr Ala Ser Ser Val Thr Phe Thr Asp He Ala Ser 85 90 95 Leu Asn He Gin Leu Thr Cys Asn He Leu Thr Phe Gly Gin Leu Glu 100 105 110 Gin Asn Val Tyr Gly He Thr He He Ser Gly Leu Pro Pro Glu Lys 1.15 120 125 Pro Lys Asn Leu Ser Cys He Val Asn Glu Gly Lys Lys Met Arg Cys 130 135 140 Glu Trp Asp Gly Gly Arg Glu Thr His Leu Glu Thr Asn Phe Thr Leu 145 150 155 160 Lys Ser Glu Trp Ala Thr His Lys Phe Ala Asp Cys Lys Ala Lys Arg 165 170 175 Asp Thr Pro Thr Ser Cys Thr Val Asp Tyr Ser Thr Val Tyr Phe Val 180 185 190 Asn He Glu Val Trp Val Glu Ala Glu Asn Ala Leu Gly Lys Val Thr 195 200 205 Ser Asp His He Asn Phe Asp Pro Val Tyr Lys Val Lys Pro Asn Pro 210 215 220 Pro His Asn Leu Ser Val He Asn Ser Glu Glu Leu Ser Ser He Leu 225 230 235 240 Lys Leu Thr Trp Thr Asn Pro Ser He Lys Ser Val He He Leu Lys 245 250 255 Tyr Asn He Gin Tyr Arg Thr Lys Asp Ala Ser Thr Trp Ser Gin He 260 265 270 Pro Pro Glu Asp Thr Ala Ser Thr Arg Ser Ser Phe Thr Val Gin Asp 275 280 285 Leu Lys Pro Phe Thr Glu Tyr Val Phe Arg He Arg Cys Met Lys Glu 290 295 300 Asp Gly Lys Gly Tyr Trp Ser Asp Trp Ser Glu Glu Ala Ser Gly He 305 310 315 320 Thr Tyr Glu Asp Arg Pro Ser Lys Ala Pro Ser Phe Trp Tyr Lys He 325 330 335 Asp Pro Ser His Thr Gin Gly Tyr Arg Thr Val Gin Leu Val Trp Lys 340 345 350 Thr Leu Pro Pro Phe Glu Ala Asn Gly Lys He Leu Asp Tyr Glu Val 355 360 365 Thr Leu Thr Arg Trp Lys Ser His Leu Gin Asn Tyr Thr Val Asn Ala 370 375 380 Thr Lys Leu Thr Val Asn Leu Thr Asn Asp Arg Tyr Leu Ala Thr Leu 385 390 395 400 Thr Val Arg Asn Leu Val Gly Lys Ser Asp Ala Ala Val Leu Thr He 405 410 415 Pro Ala Cys Asp Phe Gin Ala Thr His Pro Val Met Asp Leu Lys Ala 420 425 430 Phe Pro Lys Asp Asn Met Leu Trp Val Glu Trp Thr Thr Pro Arg Glu 435 440 445 Ser Val Lys Lys Tyr He Leu Glu Trp Cys Val Leu Ser Asp Lys Ala 450 455 460 Pro Cys He Thr Asp Trp Gin Gin Glu Asp Gly Thr Val His Arg Thr 465 470 475 480 Tyr Leu Arg Gly Asn Leu Ala Glu Ser Lys Cys Tyr Leu He Thr Val 485 490 495 Thr Pro Val Tyr Ala Asp Gly Pro Gly Ser Pro Glu Ser He Lys Ala 500 505 510 Tyr Leu Lys Gin Ala Pro Pro Ser Lys Gly Pro Thr Val Arg Thr Lys 515 520 525 Lys Val Gly Lys Asn Glu Ala Val Leu Glu Trp Asp Gin Leu Pro Val 530 535 540 Asp Val Gin Asn Gly Phe He Arg Asn Tyr Thr He Phe Tyr Arg Thr 545 550 555 560 He He Gly Asn Glu Thr Ala Val Asn Val Asp Ser Ser His Thr Glu 565 570 575 Tyr Thr Leu Ser Ser Leu Thr Ser Asp Thr Leu Tyr Met Val Arg Met 580 585 590 Ala Ala Tyr Thr Asp Glu Gly Gly Lys Asp Gly Pro Glu Phe Thr Phe 595 600 " 605 Thr Thr Pro Lys Phe Ala Gin Gly Glu He Glu Ala He Val Val Pro 610 615 620 Val Cys Leu Ala Phe Leu Leu Thr Thr Leu Leu Gly Val Leu Phe Cys 625 630 635 640 Phe Asn Lys Arg Asp Leu He Lys Lys His He Trp Pro Asn Val Pro 645 650 655 Asp Pro Ser Lys Ser His He Ala Gin Trp Ser Pro His Thr Pro Pro 660 665 670 Arg His Asn Phe Asn Ser Lys Asp Gin Met Tyr Ser Asp Gly Asn Phe 675 680 685 Thr Asp Val Ser Val Val Glu He Glu Ala Asn Asp Lys Lys Pro Phe 690 695 700 Pro Glu Asp Leu Lys Ser Leu Asp Leu Phe Lys Lys Glu Lys He Asn 705 710 715 720 Thr Glu Gly His Ser Ser Gly He Gly Gly Ser Ser Cys Met Ser Ser 725 730 735 Ser Arg Pro Ser He Ser Ser Ser Asp Glu Asn Glu Ser Ser Gin Asn 740 745 750 Thr Ser Ser Thr Val Gin Tyr Ser Thr Val Val His Ser Gly Tyr Arg 755 760 765 His Gin Val Pro Ser Val Gin Val Phe Ser Arg Ser Glu Ser Thr Gin 770 775 780 Pro Leu Leu Asp Ser Glu Glu Arg Pro Glu Asp Leu Gin Leu Val Asp 785 790 795 800 His Val Asp Gly Gly Asp Gly He Leu Pro Arg Gin Gin Tyr Phe Lys 805 810 815 Gin Asn Cys Ser Gin His Glu Ser Ser Pro Asp He Ser His Phe Glu 820 825 830 Arg Ser Lys Gin Val Ser Ser Val Asn Glu Glu Asp Phe Val Arg Leu 835 840 845 Lys Gin Gin He Ser Asp His He Ser Gin Ser Cys Gly Ser Gly Gin 850 855 860 Met Lys Met Phe Gin Glu Val Ser Ala Ala Asp Ala Phe Gly Pro Gly 865 870 875 880 Thr Glu Gly Gin Val Glu Arg Phe Glu Thr Val Gly Met Glu Ala Ala 885 890 895 Thr Asp Glu Gly Met Pro Lys Ser Tyr Leu Pro Gin Thr Val Arg Gin 900 905 910 Gly Gly Tyr Met Pro Gin 915 <210> 8 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic <400> 8 Glu Lys Arg Glu Ala Glu Ala Tyr 1 5 <210> 9 <211> 13 <212> PRT <213> Artificial Sequence <220> ■=-223 > Description of Artificial Sequence. Synthetic <400> 9 Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Val Glu 1 5 10

Claims: CLAIMS We claim: 1. A method, comprising: a) providing i) a female mammal that has not been treated with exogenous estrogen, said female to be implanted with at least one embryo, and ii) an estrogen-free composition, comprising a cytokine capable of binding to a receptor complex comprising a member of the hematopoietic cytokine receptor family; and b) administering said estrogen-free composition to said female under conditions such that said embryo implants. 2. The method of Claim 1. wherein said female mammal is a human. 3. The method of Claim 1. wherein said embryo is an in vitro fertilized embryo. 4. The method of Claim 1, wherein said cytokine is LIF. 5. A method, comprising: a) providing i) a female that has not been treated with exogenous estrogen, said female to be implanted with at least one embryo, and ii) an estrogen- free composition, comprising a ligand capable of binding to gpl 30; and b) administering said estrogen-free composition to said female under conditions such that said embryo implants. 6. The method of Claim 1, wherein said female mammal is a human. 7. The method of Claim 1, wherein said embryo is an in vitro fertilized embrvo. 8. The method of Claim 5, wherein said ligand comprises at least a portion of the receptor for IL-6.

Inventors: CHEN JIM-RAY, HERNANDEZ LIDIA, SHATZER TERESA, STEWART COLIN L, SULLIVAN TERESA, CHEN, JIM-RAY, HERNANDEZ, LIDIA, SHATZER, TERESA, STEWART, COLIN, L., SULLIVAN, TERESA
Applicants: CHEN JIM RAY, HERNANDEZ LIDIA, SHATZER TERESA, STEWART COLIN L, SULLIVAN TERESA, US HEALTH, CHEN, JIM-RAY, HERNANDEZ, LIDIA, SHATZER, TERESA, STEWART, COLIN, L., SULLIVAN, TERESA, THE GOVERNMENT OF THE UNITED STATES OF AMERICA AS REPRESENTED BY THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES
Application year: 1999
Main classification: A61K38
IPC codes: A61K38, A61K45, A61P15, A61P43