Methods and kits for maintaining pregnancy, treating follicular cysts, and synchronizing ovulation using luteinizing hormone

Abstract

This invention provides methods and kits for maintaining pregnancy in mammals. Pregnancy is maintained by administration of effective amounts of luteinizing hormone (LH) or chorionic gonadotropin (CG). Both LH and CG may be used alone, in combination with each other, or in combination with growth hormone (GH) or follicle stimulating hormone (FSH). Hormones are administered on about Day 4 to about Day 7 following insemination. Effective amounts of LH range from about 10 micrograms to about 25 milligrams, preferably about 2 to about 8 mg, and of CG range from about 100 IU (international units) to about 2000 IU. Mammals treatable by the methods of this invention include ungulates and related mammals, including bovines. Kits provided by this invention include effective amounts of one or more hormones, a device for administering the hormone(s) and instructions. This invention also provides methods for treating follicular cysts and for synchronizing ovulation in mammals using LH.

Claims

1. A method for maintaining one or more pregnancies by inducing accessory corpus luteum formation in one or more mammals, wherein insemination has been performed on said mammals and said mammals are bovine, said method comprising administering from about 25 micrograms to about 1 milligram of single chain recombinant bovine luteinizing hormone to said mammals on about Day 4 to about Day 7 after insemination, wherein administration of said single chain recombinant bovine luteinizing hormone induces accessory corpus luteum formation.

2. The method of claim 1 wherein pregnancies are maintained in more than about 40% of said mammals.

3. The method of claim 1 wherein pregnancies are maintained in more than about 50% of said mammals.

4. The method of claim 1 wherein said effective amount is between about 25 micrograms and about 250 micrograms.

5. The method of claim 1 wherein said effective amount is between about 75 micrograms and about 175 micrograms.

6. The method of claim 1 comprising administering said luteinizing hormone on about Day 4 to about Day 5 after said insemination.

7. The method of claim 1 wherein said pregnancy is maintained at about Day 15 after said insemination.

8. The method of claim 1 wherein said pregnancy is maintained at about Day 30 after said insemination.

9. The method of claim 1 wherein said pregnancy is maintained at about Day 56 after said insemination.

10. The method of claim 1 wherein administration is intramuscular.

11. The method of claim 1, comprising administering from about 75 micrograms to about 1 milligram bovine luteinizing hormone to said one or more mammals on about Day 4 to about Day 5 after said insemination.

12. The method of claim 11 comprising administering from about 75 micrograms to about 175 micrograms bovine luteinizing hormone to said one or more mammals on about Day 4 to about Day 5 after said insemination.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 illustrates a timeline for an MGA/GnRH/PGF.sub.2estrus synchronization system.

(2) FIG. 2 illustrates a timeline for a Select Synch intramuscular GnRH injection estrus synchronization system.

(3) FIG. 3 illustrates a timeline for a Co-Synch estrus or ovulation synchronization system.

(4) FIG. 4 illustrates a timeline for a Ov-Synch estrus synchronization system.

DETAILED DESCRIPTION OF THE INVENTION

(5) As used herein, ready for breeding refers to an animal that is not pregnant. The animal may have been monitored for estrus and had estrus detected. The animal may have had estrus forced.

(6) As used herein, cow refers to female bovines, including heifers.

(7) As used herein, first estrus cycle refers to the estrus cycle after insemination. In cows the first estrus cycle is about 21 days following a previous estrus.

(8) As used herein, estrus refers to the period during which an animal is most likely to become pregnant.

(9) As used herein, in heat refers to being in the time of estrus, when an animal is most sexually receptive. In cows this period lasts about 12-18 hours.

(10) As used herein, behavioral estrus refers to the behavioral demonstration that an animal is in heat, including showing standing heat.

(11) As used herein, standing heat refers to the period during which a cow is receptive to a bull and will stand to be bred or stand to be mounted by other cows.

(12) As used herein, Day 0 is the day that an animal is in behavioral estrus or the day of breeding.

(13) As used herein, forcing estrus refers to methods known in the art for forcing heat. Forcing estrus can include waiting periods, as appropriate.

(14) As used herein, forcing ovulation refers to inducing ovulation, generally within one day of the treatment used to induce ovulation.

(15) As used herein, open refers to an animal that is not pregnant.

(16) As used herein, cycling refers to an animal that is experiencing an estrus cycle, i.e., is not pregnant.

(17) As used herein, readiness for breeding refers to a time in the estrus cycle when breeding is most likely to result in pregnancy.

(18) As used herein, breeding refers to methods known in the art that pertain to making a female animal pregnant. Such methods include natural and artificial insemination. Breeding methods may include a waiting time after observation of behavioral estrus or after forcing estrus. In cattle, the waiting time after observing behavioral estrus is 12-18 hours. In cattle, after forcing estrus with prostaglandin on Day 17, the waiting time is 72-80 hours. After the last hormone injection used in forcing ovulation, breeding should within about 24 hours, e.g., between about 0 to about 24 hours, and preferably about 16 hours after that injection, so that breeding will be done before ovulation occurs.

(19) As used herein, antibody specific to refers to antibody that does not bind significantly to any sample components other than the desired component.

(20) As used herein, pregnancy testing refers to testing for pregnancy and/or non-pregnancy.

(21) As used herein, whole blood refers to blood as drawn. Whole blood contains a substantial amount of cells.

(22) As used herein, plasma refers to blood with no substantial amount of cells. Plasma does contain clotting factors.

(23) As used herein, serum refers to blood without a substantial amount of cells or clotting factors.

(24) As used herein, estrus synchronization refers to a process whereby estrus for a group of animals is forced, such that each animal is likely to be in estrus within about a 2-5-day window.

(25) As used herein ovulation synchronization refers to a process whereby ovulation for a group of animals is forced, such that each animal is likely to ovulate within a 3-4-day window.

(26) As used herein, estrus presynchronization or ovulation presynchronization refers to a process whereby the estrus cycle, often for a group of animals, is blocked or forced into a particular stage of the cycle, so that estrus or ovulation synchronization procedures that are to be performed afterwards are more successful.

(27) As used herein, cowside refers to an environment in which a domesticated animal is found, particularly in contrast to a laboratory environment.

(28) As used herein, breeding cycle time refers to the time between one breeding of an animal and the next breeding during the next estrus cycle of the same animal.

(29) As used herein, pregnant mammal refers to a mammal that has been inseminated and may be pregnant or to a plurality of inseminated mammals, some of which are likely to be pregnant.

(30) As used herein, maintaining pregnancy refers to increasing the likelihood that an animal which has been inseminated will test positive for pregnancy of will deliver a live calf or increasing likelihood that a plurality of animals that have been inseminated will test positive for pregnancy or will deliver a live calf.

(31) As used herein, effective amount refers to an amount of that is effective to produce the desired outcome.

(32) As used herein, administering refers to any method of administering known in the art that produces that desired outcome. Examples of administering include but are not limited to injecting subcutaneously, intramuscularly and intravenously.

(33) As used herein, about 98% pure refers to purity as measured by any method known in the art, including but not limited to protein electrophoresis.

(34) As used herein, insemination refers to introducing semen by any method known in the art, including, but not limited to, natural and artificial insemination.

(35) As used herein, increasing likelihood of conception refers to increasing the likelihood of detectable conception. For example, conception can be detected in bovines as early as about Day 15 after insemination by the presence of interferon-tau induced proteins.

(36) As used herein, decreasing the likelihood of embryo loss refers to decreasing the chance that an inseminated mammal will test negative for pregnancy. As used herein, decreasing the percentage of embryonic loss with respect to a plurality of mammals that have been inseminated refers to decreasing the percentage of such animals that will test negative for pregnancy.

(37) Baculovirus expression systems are well known in the art (O'Reilly et al. (1994) Baculovirus Expression Vectors: A Laboratory Manual, Oxford University Press).

(38) There are many advantages to using baculovirus for heterologous gene expression. Heterologous cDNA is expressed well. Proper transcriptional processing of genes with introns occurs but expression is less efficient. As with other eukaryotic expression systems, baculovirus expression of heterologous genes permits folding, post-translational modification and oligomerization in manners that are often identical to those that occur in mammalian cells. The insect cytoplasmic environment allows proper folding and SS bond formation, unlike the reducing environment of the E. coli cytoplasm. Post-translational processing identical to that of mammalian cells has been reported for many proteins. These include proper proteolysis, N- and O-glycosylation, acylation, amidation, carboxymethylation, phosphorylation, and prenylation. Proteins may be secreted from cells or targeted to different subcellular locations. Single polypeptide, dimeric and trimeric proteins have been expressed in baculoviruses. Finally, expression of heterologous proteins is under the control of the strong polyhedron promoter, allowing levels of expression of up to 30% of the total cell protein.

(39) SF-9, SF-21, and High-Five insect cells are commonly used for baculovirus expression. SF-9 and SF-21 are ovarian cell lines from Spodoptera frugiperda. They are grown in Grace's (or a similar) media supplemented with 10% fetal calf serum, lactalbumin, and yeastolate. High-Five cells are egg cells from Trichoplusia ni. These cells are less expensive to maintain since they may be grown without fetal calf serum. They reportedly express higher levels of recombinant proteins, although we have found these differences to be minimal. All three cell lines may be grown at room temperature (optimum=25-27 C.), and do not require CO.sub.2 incubators. Their doubling time is 18-24 hours. Expressed proteins can be recovered using protein purification methods known in the art, including use of fusion protein technology, immunoaffinity chromatography, and size-exclusion chromatography.

(40) DNA constructs prepared for introduction into a prokaryotic or eukaryotic host will typically comprise a replication system (i.e. vector) recognized by the host, including the intended DNA fragment encoding the desired polypeptide, and will preferably also include transcription and translational initiation regulatory sequences operably linked to the polypeptide-encoding segment. Expression systems (expression vectors) may include, for example, an origin of replication or autonomously replicating sequence (ARS) and expression control sequences, a promoter, an enhancer and necessary processing information sites, such as ribosome-binding sites, RNA splice sites, polyadenylation sites, transcriptional terminator sequences, and mRNA stabilizing sequences. Signal peptides may also be included where appropriate from secreted polypeptides of the same or related species, which allow the protein to cross and/or lodge in cell membranes or be secreted from the cell.

(41) An appropriate promoter and other necessary vector sequences will be selected so as to be functional in the host. Examples of workable combinations of cell lines and expression vectors are described in Sambrook et al. (1989 Molecular Cloning, Second Edition, cold Spring Harbor Laboratory, Plainview, N.Y.; Ausubel et al. (Eds.) (1995) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York; and Metzger et al. (1988) Nature, 334: 31-36. Many useful vectors for expression in bacteria, yeast, fungal, mammalian, insect, plant or other cells are well known in the art and may be obtained such vendors as Stratagene, New England Biolabs, Promega Biotech, and others. In addition, the construct may be joined to an amplifiable gene (e.g., the dihydrofolate reductase DHFR gene) so that multiple copies of the gene may be made. For appropriate enhancer and other expression control sequences, see also Enhancers and Eukaryotic Gene Expression, Cold Spring Harbor Press, NY (1983). While such expression vectors may replicate autonomously, they may less preferably replicate by being inserted into the genome of the host cell.

(42) Expression and cloning vectors will likely contain a selectable marker, that is, a gene encoding a protein necessary for the survival or growth of a host cell transformed with the vector. Although such a marker gene may be carried on another polynucleotide sequence co-introduced into the host cell, it is most often contained on the cloning vector. Only those host cells into which the marker gene has been introduced will survive and/or grow under selective conditions. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxic substances, e.g., ampicillin, neomycin, methotrexate, etc.; (b) complement auxotrophic deficiencies; or (c) supply critical nutrients not available from complex media. The choice of the proper selectable marker will depend on the host cell; appropriate markers for different hosts are known in the art.

(43) Recombinant host cells, in the present context, are those which have been genetically modified to contain an isolated DNA molecule of the instant invention. The DNA can be introduced by any means known to the art which is appropriate for the particular type of cell, including without limitation, transformation, lipofection or electroporation.

(44) Additionally, it will be recognized by those skilled in the art that allelic variations may occur in the DNA sequences, which will not significantly change activity of the amino acid sequences of the peptides that the DNA sequences encode. All such equivalent DNA sequences are included within the scope of this invention

(45) Standard techniques for cloning, DNA isolation, amplification and purification, for enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like, and various separation techniques are those known and commonly employed by those skilled in the art. A number of standard techniques are described in Sambrook et al. (1989) Molecular Cloning, Second Edition, Cold Spring Harbor Laboratory, Plainview, N.Y.; Maniatis et al. (1982) Molecular Cloning, Cold Spring Harbor Laboratory, Plainview, N.Y.; Wu (ed.) (1993) Meth. Enzymol. 218, Part I; Wu (ed.) (1979) Meth. Enzymol. 68; Wu et al. (eds.) (1983) Meth. Enzymol. 100 and 101; Grossman and Moldave (eds.) Meth. Enzymol. 65; Miller (ed.) (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Old and Primrose (1981) Principles of Gene Manipulation, University of California Press, Berkley; Schleif and Wensink (1982) Practical Methods in Molecular Biology; Glover (ed.) (1985) DNA Cloning Vol. I and II, IRL Press, Oxford, UK; Hames and Higgins (eds.) (1985) Nucleic Acid Hybridization, IRL Press, Oxford, UK; Setlow and Hollaender (1979) Genetic Engineering: Principles and Methods, Vols. 1-4, Plenum Press, New York; and Ausubel et al. (1992) Current Protocols in Molecular Biology, Greene/Wiley, New York, N.Y. Abbreviations and nomenclature, where employed, are deemed standard in the field and commonly used in professional journals such as those cited herein.

(46) A polynucleotide is said to encode a polypeptide if, in its native state or when manipulated by methods known to those skilled in the art, it can be transcribed and/or translated to produce the polypeptide or a fragment thereof. The anti-sense strand of such a polynucleotide is also said to encode the sequence.

(47) A nucleotide sequence is operably linked when it is placed into a functional relationship with another nucleotide sequence. For instance, a promoter is operably linked to a coding sequence if the promoter effects its transcription or expression. Generally, operably linked means that the sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame. However, it is well known that certain genetic elements, such as enhancers, may be operably linked even at a distance, i.e., even if not contiguous.

(48) This invention provides methods and kits for maintaining pregnancy in mammals. Pregnancy is maintained by administration of effective amounts of luteinizing hormone (LH) or chorionic gonadotropin (CG). Both LH and CG may be used alone, in combination with each other, or in combination with growth hormone (GH) or follicle stimulating hormone (FSH). Hormones are preferably administered on about Day 4 to about Day 7 following insemination. Effective amounts of LH range from about 25 micrograms to about 25 milligrams and of CG range from about 100 IU (international units) to about 2000 IU. Mammals treatable by the methods of this invention include ungulates and related mammals, including bovines. Kits provided by this invention include effective amounts of one or more hormones, a device for administering the hormone(s) and instructions.

(49) In the practice of this invention, hormone compositions of this invention are administered to mammals that are possibly pregnant as a result of insemination. Insemination is performed by any method known in the art, including artificial insemination. Preferably insemination is performed by timed artificial insemination after synchronization of ovulation. Ovulation synchronization optionally includes ovulation presynchronization, and is preferably performed by the methods of this invention.

(50) In the practice of this invention, luteinizing hormone is administered to one or more pregnant mammals to maintain pregnancy. A pregnant mammal may be a mammal that is possibly pregnant from having been inseminated, preferably during estrus, which may not even be detectably pregnant, and a plurality of pregnant mammals may be a plurality of mammals only some of which are pregnant.

(51) In one embodiment of this invention, LH is recombinant LH. The LH may be produced in a baculovirus or mammalian or other expression system. In one embodiment, recombinant LH is recovered from the milk or egg whites of a transgenic animal. Methods of producing recombinant proteins in transgenic animals are well known and have been described in U.S. Pat. Nos. 4,873,316, 5,322,775, 6,111,165, 6,472,584 and 6,528,699 and other means known in the art.

(52) In another embodiment, LH is purified from pituitary cells or pituitary tissue. Bovine LH can be purified by methods known in the art, and purified bovine LH is available on the market (AspenBio Inc., Castle Rock, Colo., Scripps Laboratories, San Diego, Calif. and BioTrend, Cologne, Germany). Purified bovine LH is also available from the NIH National Hormone and Pituitary Program (NHPP, Torrance, Calif.). When recombinant LH is used in the practice of this invention, the recombinant LH is similar structurally and has activity similar to native, purified LH. Recombinant LH can be made using cloned and mutated LH genes that encode peptides identical to native LH, or having at least about 80% homology thereto, more preferably having at least about 90% homology thereto, and most preferably having at least about 95% homology thereto and also being able to induce ovulation in a mammal. Recombinant LH made can also be made using cloned and mutated LH genes that encode peptides that are not identical to native LH, of the selected species, providing that the recombinant LH produced has a similar activity as native LH.

(53) Recombinant LH can also be made in accordance with the methods known to the art, e.g., as described in US Patent Application Publication No. 20030059898 assigned to Genzyme by Beck et al., and U.S. Pat. Nos. 6,635,256, 6,242,580, 6,238,890, 6,225,449, 6,103,501, 6,028,177, 5,985,611, 5,958,737, 5,883,073, 5,792,460, 5,759,818, 5,733,735, 5,712,122, 5,705,478, 5,585,345, 5,405,945, 5,338,835 and 5,177,193, and U.S. Patent Publication Numbers 20020160944, and 20010007757, and other means known to the art.

(54) In the practice of this invention, chorionic gonadotropin hormone is from primates, including humans, and horses. Hormones from the species to be treated, as well as hormones from other species are useful in the practice of this invention. Use of hormones from the species to be treated is less likely to cause an immune response in the treated mammal.

(55) In an embodiment of this invention, growth hormone is bovine somatotrophin (bST). In an embodiment of this invention, FSH is human FSH or bovine FSH.

(56) In the practice of this invention, pregnancy is tested by any pregnancy test known in the art, including by ultrasound or by testing for pregnancy-indicating molecules, at times appropriate for the selected species, as is known in the art. When the treated animal is a bovine, pregnancy can be tested by testing for the presence of interferon-tau induced proteins at about Day 15 (U.S. Patent Application Nos. 60/377,987; 60/377,166; 60/380,043; 60/377,921; 60/377,165; 60/377,355; 60/377,829; and 60/380,042), and/or by ultrasound at about Days 28, 45, or 56.

(57) In the practice of this invention, if a mammal is determined to not be pregnant after practicing the methods of this invention, the next cycle of estrus can be forced by methods known in the art.

(58) The methods of this invention are useful in mammals that are in estrus for the first time, have been in estrus more than once, have never had offspring, have had one or more offspring, have never been administered a hormone composition of this invention for maintaining pregnancy, or have been previously administered a hormone composition of this invention for maintaining pregnancy. The methods of this invention are specifically useful on mammals that have previously been administered hormone compositions of this invention for the purpose of maintaining pregnancy.

(59) In an embodiment of this invention, bovine LH is administered to a pregnant bovine, or a plurality of pregnant bovines, on about Day 4 to about Day 7 after insemination. In an embodiment of this invention, bovine LH is administered to a pregnant bovine, or a plurality of pregnant bovines, on about Day 4 to about Day 5 after insemination. In an embodiment of this invention, bovine LH is administered to a pregnant bovine, or a plurality of pregnant bovines, on about Day 2 to about Day 10 after insemination.

(60) In an embodiment of this invention, pregnant mammals may be screened by ultrasound for the presence of a sufficiently mature follicle before administration of a hormone composition of this invention. In an embodiment of this invention, a mature follicle of a bovine is at least about 10 mm in diameter. In an embodiment of this invention, after administration of LH, ultrasound is performed on the mammal to screen for ovulation and luteinization, (corpus luteum production).

(61) In an embodiment of this invention, bovine LH is administered in an amount ranging from about 10 micrograms to about 25 milligrams, from about 25 micrograms to about 5 milligrams, from about 25 micrograms to about 1 milligram, from about 25 micrograms to about 250 micrograms, from about 25 micrograms to about 175 micrograms, or from about 25 micrograms to about 100 micrograms, or from about 25 micrograms to about 75 micrograms. An average cow weighs about 1,000 to about 1,500 pounds. In an embodiment of this invention, bovine LH is administered in an amount that equates to about 10 nanograms to about 25 micrograms per pound of cow.

(62) In an embodiment of this invention, human CG is administered in an amount ranging from about 100 IU to about 2000 IU, from about 100 IU to about 1750 IU, or from about 250 IU to about 1000 IU.

(63) In an embodiment of this invention, the hormone composition that is to be administered is about 90% pure, about 95% pure, about 98% pure, about 99% pure, or about 100% pure, as determined by a protein purification assay known in the art.

(64) Mammals treatable by the methods of this invention include ungulates and related mammals. Mammals treatable by the methods of this invention include, but are not limited to cattle, sheep, goats, yaks, water buffaloes, bison, antelopes, gazelles, elk, reindeer, moose, bighorn sheep, giraffes, and camelids including bactrian and dromedary camels, llamas, swine, horses, alpacas, and vicunas.

(65) Preferably a hormone composition of this invention is derived from the same species as the species of mammal to be administered with said composition. If the mammal is cattle, preferably the hormone composition to be administered in the practice of this invention comprises one or more hormones that are all derived from cattle or from cattle genes.

(66) In an embodiment of this invention, a hormone composition to be administered also contains other components useful for injection as known in the art. Other components useful for injection include, but are not limited to, adjuvant and saline.

(67) In an embodiment of this invention, a hormone composition of this invention is administered more than once after insemination. In an embodiment of this invention, more than one hormone composition of this invention is administered.

(68) In the practice of this invention, pregnancy is maintained in at least about forty percent, at least about forty-two percent, at least about forty-five percent, at least about fifty percent, at least about fifty-five percent, or at least about sixty percent of cows treated using the compositions and methods of this invention.

(69) Although applicant does not wish to be bound by a particular theory, administration after insemination of hCG and/or bLH may act by inducing accessory corpus luteum formation which increases the secretion of progesterone and increases serum progesterone concentrations during the critical time when the uterus must recognize the pregnancy, resulting in increased maintenance of pregnancies.

(70) Untreated follicular cysts in an open animal can prevent an animal from cycling normally. This invention also provides a method useful in the treatment of follicular cysts in mammals, most preferably, cows. At least about 2 mg up to about 10 mg of luteinizing hormone should be injected (preferably intramuscularly) into a cow in which a follicular cyst has been diagnosed. Those of skill in the art are able to optimize dosages based on animal size, and the teachings hereof without undue experimentation. Successful resolution of the cyst may be confirmed by ultrasound or other means known to the art. If the animal is pregnant and has a normal corpus luteum in the presence of the cyst, treatment is generally not necessary.

(71) This invention also provides methods for synchronizing ovulation in a plurality of female animals. A number of techniques are known for synchronizing estrus, many of which call for the use of gonadotropin releasing hormone (GnRH), a hormone which is secreted from the hypothalamus and affects the anterior pituitary. This invention involves substituting GNRH with luteinizing hormone. The LH provides more effective synchronization than GNRH. The LH is administered in effective amounts, preferably in amounts between about 2 mg and about 10 mg in cattle. One skilled in the art can optimize dosages based on animal size and response without undue experimentation.

(72) One estrus synchronization system known to the art is the MGA/GnRH/PGF.sub.2 System (Wood, S. L., et al. (2001), Improved synchrony of estrus and ovulation with the addition of GNRH to a melengestrol acetate-prostaglandin synchronization treatment in beef heifers, J. Anim. Sci. 79:2210-2216). Melengestrol acetate (MGA) is an orally active synthetic progestin that was developed to control estrus in feedlot heifers (Lauderdale, et al., 1977). This progestin can be used in estrous synchronization to mimic progesterone and can stimulate estrus in heifers. Since small amounts of MGA are used, be careful when mixing bulk rations to ensure even distribution throughout the ration. PGF.sub.2 is prostaglandin, a hormone released from the uterus once the female recognizes she is not pregnant. Prostaglandin causes the CL to regress or diminish and, once this occurs, progesterone concentrations decrease rapidly.

(73) The MGA/GnRH/PGF.sub.2 system includes the following steps: Feed MGA for 14 days at 0.5 mg/hd/d. On day 26, inject a shot of GNRH IM; follow this 7 days later (day 33) by a shot of prostaglandin IM. Heat check and breed from day 33 to day 38 (see FIG. 1).

(74) In the system of this invention, used for ovulation synchronization, LH in the above-described dosage is administered instead of GNRH. If desired, the animal can be bred immediately after this treatment. In this invention, progestin may be substituted for MGA. MGA is illegal for use in lactating dairy cows, but the method is useful for beef producers.

(75) Other estrus synchronization systems known to the art include the Select Synch system. This system comprises the following steps: Inject GNRH intramuscularly (IM) on day 0, followed by prostaglandin IM on day 7. Research by Geary and Whittier (Geary, T. W., and J. C. Whittier (1999), Various protocols for synchronization of estrus or ovulation using GNRH and prostaglandin, 1999 Beef Program Report. Department of Anim. Sci., Colorado State University) report pregnancy rates of 61 percent for cattle bred based on standing heat (see FIG. 2).

(76) Another estrus synchronization method is the Co-Synch system (Geary, T. W., and J. C. Whittier (1999), Various protocols for synchronization of estrus or ovulation using GNRH and prostaglandin, 1999 Beef Program Report. Department of Anim. Sci., Colorado State University), which involves the following steps: Inject GNRH IM on day 0, followed by prostaglandin IM on day 7. Inject GNRH again on day 9; then time breed (see FIG. 3).

(77) A further estrus synchronization system is the Ov-Synch system (Geary, T. W., and J. C. Whittier (1999), Various protocols for synchronization of estrus or ovulation using GnRH and prostaglandin, 1999 Beef Program Report. Department of Anim. Sci., Colorado State University), which involves the following steps: Inject GnRH IM on day 0, followed by prostaglandin IM on day 7. Inject GNRH again at day 9, and time breed on day 10 (see FIG. 4).

(78) Another common estrus synchronization program in dairy cattle is called Heatsynch which uses GNRH (day 0), PGF (day 7), then estradiol cipronate (ECP) (day 8). Cows are observed for heat and bred at any heat. Any cows that are not found in heat are time bred 48 hours after the ECP injection (day 10).

(79) In the systems of this invention, LH is substituted for GNRH in the dosages described above.

(80) The following examples are provided for illustrative purposes, and are not intended to limit the scope of the invention as claimed herein. Any variations in the exemplified articles that occur to the skilled artisan are intended to fall within the scope of the present invention.

EXAMPLES

Example 1

(81) Expression of Recombinant bLH in a Baculovirus Expression System

(82) DNA sequences encoding the alpha and beta bovine luteinizing hormone subunits are ligated into a bacterial transfer vector. Inserts for protein purification are optionally included. The insert is flanked by portions of viral genes to permit homologous recombination with replication defective, linear, viral DNA. The direction of the inserts relative to the polyhedron promoter is verified. The sequences of the inserts are optionally verified. Plasmids are purified for transfection into insect cells.

(83) Insect cells are cotransfected with the alpha and beta LH recombinant transfer vectors and linearized viral vector. Optionally, alpha and beta LH transfections are performed individually. Transfection and transformation methods are well known in the art and include electroporation, lipid, and calcium phosphate-mediated transformation methods. Replicative viruses form by intracellular homologous recombination between the ends of the viral molecules and portions of the transfer vector flanking alpha and beta LH. Alpha and beta bovine LH are inserted into the virus and complement defective viral gene(s) to permit viral replication. Marker proteins are optionally included and expressed upon selected insertion events.

(84) The transfected cell supernatants are harvested. Insect cells are infected with dilutions of the supernatant to isolate single virus plaques that optionally express marker proteins. Additional insect cells are infected with virus from selected plaques to amplify the quantity and titer of viral stocks. Protein expression is optionally examined by Western blotting. Protein expression and activity is tested by bioassay.

Example 2

(85) Estrus Synchronization and Artificial Insemination Control

(86) Several hundred cows were induced to ovulate using a standard estrus synchronization protocol, artificially inseminated, and intravenously administered sterile water.

(87) On Days 14 and 21 serum was collected for progesterone assay. These collection times represent the critical period for progesterone to maintain pregnancy, and the expected time of lowest progesterone concentration if the animal is not pregnant and returns to estrus. Ultrasound examination of the ovarian structures was performed on Days 14 and 21 to evaluate correlation of CL size and progesterone concentration, and to allow comparison of both measurements with treatment and with outcome of the breeding. On post insemination Days 28, 35, and 56, ultrasound examination of the uterus was used to determine pregnancy status. Day 28 is the earliest time for reliably visualizing a pregnancy. Pregnancies were closely monitored at these times to determine rates of embryonic loss and final outcome of each breeding.

(88) Data was analyzed by statistical software (SAS). Herd and season effects were analyzed using ANOVA and regression analysis. At Day 56, 38% of treated cows were pregnant.

Example 3

(89) Administration of 1660 IU hCG on Day 7 After Insemination

(90) On a weekly basis, cows at the end of the elective waiting period were induced to ovulate using a standard estrus synchronization protocol, and at the time of breeding each cow was assigned to either the treatment or control group (Example 2) on an alternating basis. On Day 7, post-breeding cows were intramuscularly administered 1,660 IU hCG. (The World Health Organization standard for hCG activity has been determined to be approximately 10,000 IU per milligram.)

(91) On Days 14 and 21 serum was collected for progesterone assay. These collection times represent the critical period for progesterone to maintain pregnancy, and the expected time of lowest progesterone concentration if the animal is not pregnant and returns to estrus. Ultrasound examination of the ovarian structures was performed on Days 14 and 21 to evaluate correlation of CL size and progesterone concentration, and to allow comparison of both measurements with treatment and with outcome of the breeding. On post insemination Days 28, 35, and 56, ultrasound examination of the uterus was used to determine pregnancy status. Day 28 is the earliest time for reliably visualizing a pregnancy. Pregnancies were closely monitored at these times to determine rates of embryonic loss and final outcome of each breeding.

(92) Several hundred cows were tested. Data was analyzed by statistical software (SAS). Herd and season effects were analyzed using ANOVA and regression analysis. At Day 56, 50% of treated cows were pregnant.

Example 4

(93) Administration of 830 IU hCG on Day 7 After Insemination

(94) Several hundred cows at the end of the elective waiting period were induced to ovulate using a standard estrus synchronization protocol, and at the time of breeding each cow was assigned to either the treatment or control group (Example 2) on an alternating basis. On Day 7, post-breeding cows were intramuscularly administered 1,660 IU hCG.

(95) On Days 14 and 21 serum was collected for progesterone assay. Ultrasound examination of the ovarian structures was performed on Days 14 and 21 to evaluate correlation of CL size and progesterone concentration, and to allow comparison of both measurements with treatment and with outcome of the breeding. On post insemination Days 28, 35, and 56, ultrasound examination of the uterus was used to determine pregnancy status. Pregnancies were closely monitored at these times to determine rates of embryonic loss and final outcome of each breeding.

(96) Several hundred cows were tested. Data was analyzed by statistical software (SAS). Herd and season effects were analyzed using ANOVA and regression analysis. At Day 56, 52% of treated cows were pregnant.

Example 5

(97) Administration of 830 IU hCG on Day 5 After Insemination

(98) Cows are forced into estrus, artificially inseminated, and intramuscularly administered 830 IU hCG on Day 5 after insemination. Serum is collected on Days 14 and 21 and assayed for progesterone. Ultrasound is performed on Days 14 and 21. Ultrasound examination is performed on Days 28, 35, and 56. At Day 56, at least about 50% of cows are pregnant.

Example 6

(99) Administration of 156, 83, 25 and 10 Micrograms and 1, 10 and 25 mg bLH on Day 5 After Insemination

(100) Cows are forced into estrus, artificially inseminated, and intramuscularly administered 156, 83, 25 and 10 micrograms or 1, 10 and 25 mg bLH, purified from bovine pituitary, on Day 5 after insemination. Serum is collected on Days 14 and 21 and assayed for progesterone. Ultrasound is performed on Days 14 and 21. Ultrasound examination is performed on Days 28, 35, and 56. At Day 56, improvement in maintenance of pregnancy is shown compared to cows not administered bLH or other hormone, and cows administered hCG only.

Example 7

(101) Administration of 156, 83, 25 and 10 Micrograms and 1, 10 and 25 mg bLH on Day 5 After Insemination

(102) Cows are forced into estrus, artificially inseminated, and intramuscularly administered 156, 83, 25 and 10 micrograms or 1, 10 and 25 mg recombinant bLH, obtained from a baculovirus expression system (Example 1), on Day 5 after insemination. Serum is collected on Days 14 and 21 and assayed for progesterone. Ultrasound is performed on Days 14 and 21. Ultrasound examination is performed on Days 28, 35, and 56. At Day 56, improvement in maintenance of pregnancy is shown compared to cows not administered bLH or other hormone, and cows administered hCG only.

Example 8

(103) Intravenous Administration of 10 Micrograms bLH on Day 5 After Insemination

(104) Cows are forced into estrus, artificially inseminated, and intravenously administered 10 micrograms bLH, purified from bovine pituitary, on Day 5 after insemination. Serum is collected on Days 14 and 21 and assayed for progesterone. Ultrasound is performed on Days 14 and 21. Ultrasound examination is performed on Days 28, 35, and 56. At Day 56, improvement in maintenance of pregnancy is shown compared to cows not administered bLH or other hormone, and cows administered hCG only.

Example 9

(105) Development of Accessory Corpus Lutea

(106) Tests were performed over a three-month period utilizing a total of 31 cows in eight different tests. Bovine luteinizing hormone was administered in varying dosage levels, from 0.5 mg to 8 mg in sterile saline to cows four days after artificial insemination. Tests determined that an effective dosage level was 2 mg, intravenously, or greater. Using this 2 mg dosage in a limited trial of 15 cows, we were able to create at least one accessory corpus luteum in twelve cows (80% response). Ovaries were examined by ultrasound both before and after treatment using a Sonosite VET180plus with an 11 mm broadband sector transducer with a range of 4-7 mHz. The after-treatment scan was performed seven days after injection. Results are shown in Table 1.

(107) TABLE-US-00001 TABLE 1 PRODUCTION OF ACCESSORY CORPUS LUTEUM BY INJECTION OF BOVINE LUTEINIZING HORMONE Dose Number Number Percent (mg) Responding* Treated Responding 0.5 0 1 0 1 0 3 0 2 12 15 80 3 5 5 100 4 3 3 100 6 2 2 100 7 1 1 100 8 1 1 100 *Response = development of at least one accessory corpus luteum.
This response is comparable to the accessory corpus luteum formation found after administering a dose of human chorionic gonadotropin (hCG) to lactating dairy cattle.

Example 10

(108) Treatment of Follicular Cysts in Cattle

(109) Three non-pregnant, non-inseminated cows were examined by ultrasound to determine the presence of follicular cysts. The cows were treated with injections of 2 mg luteinizing hormone in saline. Seven more non-pregnant, non-inseminated cows diagnosed with follicular cysts were treated as above. Forty-eight hours after treatment, the cows are examined for the presence of cysts using ultrasound to confirm that the cysts have resolved.

Example 11

(110) Ovulation Synchronization

(111) Ten non-pregnant cows not exhibiting signs of being in heat are injected with 2 mg luteinizing hormone, IM. Seven days later, the cows are injected IM with 25 mg prostaglandin. 9 days later, the cows are again injected with 2 mg of luteinizing hormone. The cows are artificially inseminated within a few hours after the second injection of luteinizing hormone, resulting in a pregnancy rate greater than about forty percent. Compared with the use of GnRH, the use of LH provides more reliable synchronization and a greater pregnancy rate.

Example 12

(112) Cloning of bLH Alpha Subunit

(113) RNA was extracted from 1 bovine pituitary gland using RNeasy Midiprep (Qiagen cat#75142). RT-PCR was performed using Superscript One-Step RT PCR with Platinum Taq (Invitrogen cat#10928-034). Primers used in this reaction were bLH alpha BamH1 U (GGATCCATGGGATTACTACAGAAA) and bLH alpha RI L (GAATTCTTAGGATTTGTGATAATAAC). RT-PCR product was gel purified using QiaQuick (Qiagen cat#28704). Purified product was polished and ligated into pCRScript cloning vector using kit reagents (Statagene cat#211188). Ligation was transformed into Top 10 electro competent E. Coli (Invitrogen cat#C4040-50) and plated onto LB agar with ampicillin. Transformants were analyzed by restriction digest using BamHI (NEB cat# R0136S) and sequence confirmed by DNA sequencing (MMR).

Example 13

(114) Cloning of bLH Beta Subunit

(115) RNA was extracted from 3 bovine pituitary glands using Tri-Reagent BD (Sigma cat# T3809). DNA was synthesized using iScript cDNA Synthesis Kit (BioRad cat#170-8890). Primary PCR was performed using the above cDNA, Deep Vent DNA Polymerase (NEB cat# M0258S) and the following primers: bLH-B L 9-9-0 (TTTCCAGAGTTAGGATGGGCATGG) and bLH-B U 9-9-03 (CAAGGATGGAGATGTTCCAGGGAC). Secondary PCR was performed using the primary PCR product as template, Deep Vent DNA Polymerase and the following primers: 5bglMEbLHb (AGATCTATGGAGATGTTCCAGGGACTG) and 3bLHbetaR1 (GAATTCAGTGGGGCATCCTTAGAGGAAGAG). Secondary PCR product was gel purified using QiaQuick and adenosine extension reaction was performed using PCR Master Mix (Promega cat# M7501). The product was ligated into pCR2.1 TOPO Cloning Vector (Invitrogen cat# K4500-01). Ligation was transformed into chemical competent Top 10F E. coli (Invitrogen cat# C3030-03) and plated onto LB agar with ampicillin. Transformants were analyzed by restriction digest using EcoRI (NEB cat# R0101S) and sequence confirmed by DNA sequencing (Lark Technologies).

Example 14

(116) Insect Expression Strategies

(117) Baculovirus Expression

(118) Bovine LH alpha and beta are inserted into pBac4x-1 (Novagen cat#70045-3) separately and together for both individual and dual expression using the BacVector (Novagen cat#70077) Baculovirus Expression system in sf9, Sf21, and High Five insect cells. Bovine LH alpha and beta are inserted into pFastBack Dual (Invitrogen cat#10712-024) for dual expression in Sf9, Sf21, and High Five insect cells.

(119) Bovine LH Alpha Into pBac4x-1

(120) bLH alpha in pCRScript and pBac4X-1 were digested with NotI (NEB cat# R0189S) and XhoI (NEB cat# R0146S). bLH alpha insert and cut vector were gel purified using QiaQuick and ligated using T4 DNA Ligase (NEB cat# M0202S). Ligation was transformed into electro competent Top10 E. coli and plated onto LB agar with ampicillin. Transformants were analyzed by restriction digest using NotI and XhoI and sequence was confirmed by DNA sequencing (Lark Technologies).

(121) Bovine LH beta into pBac4x-1

(122) bLH beta in pCR2.1 and pBac4x-1 were digested with Bgl II (NEB cat# R0144S) and EcoRI. bLH beta insert and cut vector were gel purified using QiaQuick and ligated using T4 DNA Ligase (NEB). Ligation was transformed into electro competent XLI Blue and plated onto LB agar with ampicillin. Transformants were analyzed by restriction digest using Bgl II and EcoRI and sequence was confirmed by DNA sequencing (Lark Technologies).

(123) Bovine LH Alpha and Beta Into pBac4x-1

(124) bLH alpha and bLH beta each in pBac4x-1 were cut with AgeI (NEB cat# R0552S) and Bgl II. The fragment containing the alpha insert and the fragment containing the beta subunit were gel purified using QIAex II (Qiagen cat#20021). Fragments were ligated together using T4 DNA Ligase (NEB). Ligation was transformed into chemical competent Top10 E. coli and plated onto LB agar with ampicillin. Transformants were analyzed by restriction digest using EcoRI and sequence confirmed by DNA sequencing (Lark Technologies).

(125) Insect Cell Line Expression

(126) Bovine LH alpha and beta are inserted into pIZ/V5-His (Invitrogen cat# V8000-01) and pIB/V5-His (Invitrogen cat# V8020-01) separately for co-expression using the InsectSelect System for stable cell line expression in Sf9, Sf21 and High Five insect cells. Co-expression is performed using bLH alpha/pIZ/V5-His with bLH beta/pIB/V5-His and also using bLH alpha/pIB/V5-His with bLH beta/pIZ/V5-His. Stable lines expressing single chains are also be infected with baculovirus encoding the complementary chain.

(127) Bovine LH Alpha Into pIZ/V5-His

(128) bLH alpha in pBac4X-1 and pIZ/V5-His were each digested with BamHI and EcoRI. Fragments containing bLH alpha and cut pIZ/V5-His were gel purified using QIAex II. Fragments were ligated together using T4 DNA Ligase (Invitrogen cat#15224-017). Ligation was transformed into electro competent Top10 E. coli (Invitrogen cat# C664-11) and plated onto LB agar with Zeocin. Transformants were analyzed by restriction digest with Sac (NEB cat# R0156) and EcoRI and sequence confirmed by DNA sequencing (lark Technologies).

(129) Bovine LH Beta Into pIZ/V5-His

(130) bLH beta in pCR2.1 was digested with Bgl II and EcoRI and pIZ/V5-His was digested with BamHI and EcoRI. Fragments containing bLH beta and cut pIZ/V5-His were gel purified using QIAex II. Fragments were ligated together using T4 DNA Ligase (Invitrogen). Ligation was transformed into electro competent Top10 E. coli and plated onto LB agar with Zeocin. Transformants were analyzed by restriction digest with Sac1 and EcoRI and sequence confirmed by DNA sequencing (Lark Technologies)

(131) Bovine LH Alpha Into pIB/V5-His and Bovine LH Beta Into pIB/V5-His

(132) The cloning strategy follows pIZ/V5-His with the exception that clone selection occurs using ampicillin and cell selection occurs using blasticidin.

Example 15

(133) Mammalian Expression Strategies

(134) Bovine LH alpha and beta is inserted into pBudCE4.1 (Invitrogen cat# V532-20) for dual expression in COS7, CHO, 293 and 3T3 mammalian cells. Bovine LH alpha and beta are also inserted into pBudCE4.1 and pWE1 (ATCC cat#87 678) separately for co-expression in COS7, CHO, 293 and 3T3 mammalian cells. Co-expression is performed using bLH alpha/pBudCE4.1 with bLH beta/pWE1 and also using bLH alpha/pWE1 with bLH beta/pBudCE4.1.

(135) Bovine LH alpha and beta subunits into pBudCE4.1 (Invitrogen cat# V532-20) for dual expression in COS7, CHO, 293 and 3T3 mammalian cells is as follows: bLH alpha is inserted into pBudCE4.1 using the NotI/XhoI sites. bLH beta is inserted into pBudCE4.1 using the BamH1/EcoR1 sites. Bovine LH alpha and beta is inserted into pWE1 using BamH1 and EcoR1.

Example 16

(136) Single-Chain Recombinant Bovine LH

(137) Single-chain recombinant bovine LH can be made in accordance with the methods described in U.S. Pat. No. 6,242,580, which discloses recombinant LH wherein the beta subunit is covalently linked to the alpha subunit. Alternatively, a linker is present between the beta and alpha subunits. Single-chain forms need only a single gene to be transcribed during recombinant production and are advantageous over the dimeric forms in terms of stability of the protein. SEQ ID NOs 1-4 present the nucleotide sequences for bovine LH beta and alpha subunits. Expression vectors where the C-terminus of the bovine beta subunit is linked to the N-terminus of the bovine alpha subunit are transfected into CHO cells for expression.

(138) It will be appreciated by those of ordinary skill in the art that methods, compositions, and kits other than those specifically disclosed herein are available in the art and can be employed in the practice of this invention. All art-known functional equivalents are intended to be encompassed within the scope of this invention.

(139) TABLE-US-00002 SEQ ID NO:1 X00050. Bovine mRNA for a...[gi:606] LOCUSBTPASH713 bpmRNAlinearMAM 30-MAR-1995 DEFINITION Bovine mRNA for alpha-subunit of pituitary hormones. (glycoprotein hormones). ACCESSION X00050 J00009 K00527 V01493 VERSION X00050.1 GI:606 KEYWORDS glycoprotein; hormone; signal peptide. SOURCE Bos taurus (cow) ORGANISM Bos taurus Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Cetartiodactyla; Ruminantia; Pecora; Bovoidea; Bovidae; Bovinae; Bos. REFERENCE 1 (bases 98 to 661) AUTHORS Nilson,J.H., Thomason,A.R., Cserbak,M.T., Moncman,C.L. and Woychik,R.P. TITLE Nucleotide sequence of a cDNA for the common alpha subunit of the bovine pituitary glycoprotein hormones. Conservation of nucleotides in the 3-untranslated region of bovine and human pre-alpha subunit mRNAs JOURNAL J. Biol. Chem. 258 (8), 46794682 (1983) MEDLINE 83161058 PUBMED6187740 REFERENCE 2 (bases 1 to 713) AUTHORS Erwin,C.R., Croyle,M.L., Donelson,J.E. and Maurer,R.A. TITLE Nucleotide sequence of cloned complementary deoxyribonucleic acid for the alpha subunit of bovine pituitary glycoprotein hormones JOURNAL Biochemistry 22 (20), 48564860 (1983) MEDLINE 84024633 PUBMED6688736 COMMENT Data kindly reviewed (09-MAY-1985) by R.A. Maurer. FEATURESLocation/Qualifiers source 1..713 /organism=Bos taurus /db_xref=taxon:9913 mRNA <1..713 /product=messenger RNA CDS 78..440 /note=alpha-subunit precursor /codon_start=1 /protein_id=CAA24932.1 /db_xref=GI:607 /db_xref=SWISS-PROT:P01217 /translation=MDYYRKYAAVILTILSLFLQILHSFPDGEFTMQGCPECKLKENK YFSKPDAAIYQCMGCCFSRAYPTPARSKKTMLVPKNITSEATCCVAKAFTKATVMG NV RVENHTECHCSTCYYHKS sig_peptide 78..149 /note=signal peptide mat_peptide 150..437 /product=alpha-subunit misc_feature 688..693 /note=polyA signal polyA_site 713 /note=polyadenylation site BASE COUNT 209 a164 c133 g207 t ORIGIN 1 aaaaactaaa attcttcttc agatccacag tcaactgccc tgactacatt ctgcaaaaat 61 ccagaggacg aagagccatg gattactaca gaaaatatgc agctgtcatt ctgaccattt 121 tgtctctgtt tctgcaaatt ctccattcct ttcctgatgg agagtttaca atgcagggct 181 gtcctgaatg caagctaaaa gaaaacaaat acttctccaa gccagatgct gcaatctatc 241 agtgcatggg gtgctgcttc tccagggcat accccactcc agcgaggtct aagaagacaa 301 tgttggtccc caagaacatc acctcggaag ctacatgctg tgtggccaaa gcatttacca 361 aggccacagt gatgggaaat gtcagagtgg agaaccacac cgagtgccac tgcagcactt 421 gttattatca caaatcctaa tagtttgcag tgggccttgc tgatgatggc tgacttgctc 481 aaaaggaaaa ttaatttgtc cagtgtctat ggctttgtga gataaaaccc tccttttcct 541 tgccatacca tttttaacct gctttgagaa tatactgcag ctttattgct tttctcctta 601 tcctacaata taatcagtag tcttgatctt ttcatttgga atgaaatatg gcatttagca 661 tgaccataaa aagctgattc cactggaaat aaagtctttt aaatcatcac tct // Revised: July 5, 2002. SEQ ID NO:2 M10077. Bovine lutropin (...[gi:163300] LOCUSBOVLHBX629 bpmRNAlinearMAM 27-APR-1993 DEFINITION Bovine lutropin (LH) beta subunit mRNA, complete cds. ACCESSION M10077 VERSION M10077.1 GI:163300 KEYWORDS glycoprotein; lutropin. SOURCE Bos taurus (cow) ORGANISM Bos taurus Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Cetartiodactyla; Ruminantia; Pecora; Bovoidea; Bovidae; Bovinae; Bos. REFERENCE 1 (bases 1 to 629) AUTHORS Maurer,R.A. TITLE Analysis of several bovine lutropin beta subunit cDNAs reveals heterogeneity in nucleotide sequence JOURNAL J. Biol. Chem. 260 (8), 46844687 (1985) MEDLINE 85182575 PUBMED3838746 COMMENT Original source text: Bovine pituitary lambda gt-11 library, cDNA to mRNA, clones LH[-7,-14,-8,-6]. Variations between the four clone sequences most likely reflects different processing of the precursor mRNAs [1]. Draft entry and sequence [1]in computer-readable form kindly provided by R. Maurer (04-OCT-1985). FEATURES Location/Qualifiers source 1..629 /organism=Bos taurus /db_xref=taxon:9913 mRNA <1..514 /note=LHb mRNA (clone LH-7) CDS 2..421 /note=luteinizing hormone beta subunit prepeptide /codon_start=1 /protein_id=AAA30623.1 /db_xref=GI:163301 /translation=MFQGLLLWLLLGVAGVWASRGPLRPLCQPINATLAAEKEACPVC ITFTTSICAGYCPSMKRVLPVILPPMPQRVCTYHELRFASVRLPGCPPGVDPMVSFPV ALSCHCGSCRLSSTDCGGPRTQPLACDHPPLPDILFL sig_peptide 2..55 /note=luteinizing hormone beta subunit signal peptide mat_peptide 56..418 /product=luteinizing hormone beta subunit mRNA <28..629 /note=LHb mRNA (clone LH-14) mRNA <31..514 /note=LHb mRNA (clone LH-8) mRNA <70..514 /note=LHb mRNA (clone LH-6) variation 169 /note=c in clones LH[-7,-14,-8]; t in clone LH-6 variation 178..182 /note=gaagc in clones LH[-7,-14,-6]; gc in clone LH-8 variation 329 /note=t in clone LH-7; c in clones LH[-6,-8,-14] variation 447 /note=c in clones LH[-7,-8,-6]; t in clone LH-14 BASECOUNT 127 a217 c144 g141 t ORIGIN 42 bp upstream of HpaII site. 1 gatgttccag ggactgctgc tgtggctgct gctgggcgtg gccggggtgt gggcttccag 61 ggggccactg cggccgctgt gccagcccat caacgccacc ctggcggctg agaaggaggc 121 ctgccctgtc tgtatcactt tcaccaccag catctgcgcc ggctactgcc ccagcatgaa 181 gcgggtgctg cctgtcatcc tgccgcccat gccccagcgg gtgtgcacct accatgagct 241 gcgcttcgcc tccgttcggc tccccggctg cccacctgga gtggacccaa tggtctcctt 301 ccccgtggcc ctcagctgtc actgtggatc ctgccgcctc agcagcactg actgcggggg 361 tcccagaacc caacccttgg cctgtgacca ccccccgctc ccagacatcc tcttcctcta 421 aggatgcccc acttcaacct cccatgccca tcctaactct ggaaaccagc agacactctt 481 cccctccctt cccaataaag acttctcaaa ctgcctaggc tggcctaata ataattgtaa 541 tcattattaa cccagaagtt cttcaaatat aagattaaaa agatgaacag atataattct 601 tacccttatt aaagacaaaa gagttttct SEQ. ID. NO:3 NM_173901. Bos taurus glycop...[gi:27806912] LOCUSNM_173901731 bpmRNAlinearMAM 05-OCT-2003 DEFINITION Bos taurus glycoprotein hormones, alpha polypeptide (CGA), mRNA. ACCESSION NM_173901 VERSION NM_173901.1 GI:27806912 KEYWORDS SOURCE Bostaurus(cow) ORGANISM Bos taurus Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Cetartiodactyla; Ruminantia; Pecora; Bovoidea; Bovidae; Bovinae; Bos. REFERENCE 1 (bases 1 to 731) AUTHORS Goodwin,R.G., Moncman,C.L., Rottman,F.M. and Nilson,J.H. TITLE Characterization and nucleotide sequence of the gene for the common alpha subunit of the bovine pituitary glycoprotein hormones JOURNAL Nucleic Acids Res. 11 (19), 68736882 (1983) MEDLINE 84041490 PUBMED6314263 REFERENCE 2 (bases 1 to 731) AUTHORS Erwin,C.R., Croyle,M.L., Donelson,J.E. and Maurer,R.A. TITLE Nucleotide sequence of cloned complementary deoxyribonucleic acid for the alpha subunit of bovine pituitary glycoprotein hormones JOURNAL Biochemistry 22 (20), 48564860 (1983) MEDLINE 84024633 PUBMED6688736 COMMENT PROVISIONAL REFSEQ: This record has not yet been subject to final NCBI review. The reference sequence was derived from X00003.1. FEATURESLocation/Qualifiers source 1..731 /organism=Bos taurus /mol_type=mRNA /db_xref=taxon:9913 /chromosome=9 /map=9 gene 1..731 /gene=CGA /db_xref=GeneID:280749 /db_xref=LocusID:280749 CDS 101..463 /gene=CGA /note=chorionic gonadotropin, alpha chain /codon_start=1 /product=glycoprotein hormones, alpha polypeptide /protein_id=NP_776326.1 /db_xref=GI:27806913 /db_xref=GeneID:280749 /db_xref=LocusID:280749 /translation=MDYYRKYAAVILAILSLFLQILHSFPDGEFTMQGCPECKLKENK YFSKPDAPIYQCMGCCFSRAYPTPARSKKTMLVPKNITSEATCCVAKAFTKATVMGN V RVENHTECHCSTCYYHKS sig_peptide 101..172 /gene=CGA misc_feature 173..460 /gene=CGA /note=hormone6; Region: Glycoprotein hormone /db_xref=CDD:pfam00236 mat_peptide 173..460 /gene=CGA /product=unnamed /label=pit_mat misc_feature 200..460 /gene=CGA /note=GHA; Region: Glycoprotein hormone alpha chain homologues. Also called gonadotropins. Glycoprotein hormones consist of two glycosylated chains (alpha and beta) of similar topology /db_xref=CDD:smart00067 ORIGIN 1 gcagttgctg agaaatcaca agacaaaact aaaattcttc ttcagatcca cagtcaactg 61 ccctgactac attctgcaaa aatccagagg acgaagagcc atggattact acagaaaata 121 tgcagctgtc attctggcca ttttgtctct gtttctgcaa attctccatt cctttcctga 181 tggagagttt acaatgcagg gctgtcctga atgcaagcta aaagaaaaca aatacttctc 241 caagccagat gctccaatct atcagtgcat ggggtgctgc ttctccaggg cataccccac 301 tccagcgagg tctaagaaga caatgttggt ccccaagaac atcacctcgg aagctacatg 361 ctgtgtggcc aaagcattta ccaaggccac agtgatggga aatgtcagag tggagaacca 421 caccgagtgc cactgcagca cttgttatta tcacaaatcc taatagtttg cagtgggcct 481 tgctgatgat ggctgacttg ctcaaaagga aaattaattt gtccagtgtc tatggctttg 541 tgagataaaa ccctcctttt ccttgccata ccatttttaa cctgctttga gaatatactg 601 cagctttatt gcttttctcc ttatcctaca atataatcag tagtcttgat cttttcattt 661 ggaatgaaat atggcattta gcatgaccat aaaaagctga ttccactgga aataaagtct 721 tttaaatcat c SEQ. ID. NO:4 NM_173930. Bos taurus lutein...[gi:27806854] LOCUSNM_173930426 bpmRNAlinearMAM 05-OCT-2003 DEFINITION Bos taurus luteinizing hormone beta polypeptide (LHB), mRNA. ACCESSION NM_173930 VERSION NM_173930.1 GI:27806854 KEYWORDS SOURCE Bos taurus (cow) ORGANISM Bostaurus Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Cetartiodactyla; Ruminantia; Pecora; Bovoidea; Bovidae; Bovinae; Bos. REFERENCE 1 (bases 1 to 426) AUTHORS Virgin,J.B., Silver,B.J., Thomason,A.R. and Nilson,J.H. TITLE The gene for the beta subunit of bovine luteinizing hormone encodes a gonadotropin mRNA with an unusually short 5-untranslated region JOURNAL J. Biol. Chem. 260 (11), 70727077 (1985) MEDLINE 85207729 PUBMED2987241 REFERENCE 2 (bases 1 to 426) AUTHORS Maurer,R.A. TITLE Analysis of several bovine lutropin beta subunit cDNAs reveals heterogeneity in nucleotide sequence JOURNAL J. Biol. Chem. 260 (8), 46844687 (1985) MEDLINE 85182575 PUBMED3838746 COMMENT PROVISIONAL REFSEQ: This record has not yet been subject to final NCBI review. The reference sequence was derived from M11506.1. FEATURESLocation/Qualifiers source 1..426 /organism=Bos taurus /mol_type=mRNA /db_xref=taxon:9913 /chromosome=18 /map=18 gene 1..426 /gene=LHB /db_xref=GeneID:280839 /db_xref=LocusID:280839 CDS 1..426 /gene=LHB /note=precursor /codon_start=1 /product=luteinizing hormone beta polypeptide /protein_id=NP_776355.1 /db_xref=GI:27806855 /db_xref=GeneID:280839 /db_xref=LocusID:280839 /translation=MEMFQGLLLWLLLGVAGVWASRGPLRPLCQPINATLAAEKEACP VCITFTTSICAGYCPSMKRVLPVILPPMPQRVCTYHELRFASVRLPGCPPGVDPMVSF PVALSCHCGPCRLSSTDCGGPRTQPLACDHPPLPDILFL sig_peptide 1..60 /gene=LHB mat_peptide 61..423 /gene=LHB /product=luteinizing hormone beta polypeptide misc_feature 73..393 /gene=LHB /note=GHB; Region: Glycoprotein hormone beta chain homologues. Also called gonadotropins. Glycoprotein hormones consist of two glycosylated chains (alpha and beta) of similar topology /db_xref=CDD:smart00068 variation 68 /gene=LHB /note=g in DNA; a in cDNA /replace=a variation 81..82 /gene=LHB /note=gc in DNA; cg in cDNA /replace=cg ORIGIN 1 atggagatgt tccagggact gctgctgtgg ctgctgctgg gcgtggccgg ggtgtgggct 61 tccagggggc cactgcggcc gctgtgccag cccatcaacg ccaccctggc ggctgagaag 121 gaggcctgcc ctgtctgtat cactttcacc accagcatct gcgccggcta ctgccccagc 181 atgaagcggg tgctgcctgt catcctgccg cccatgcccc agcgggtgtg cacctaccat 241 gagctgcgct tcgcctccgt tcggctcccc ggctgcccac ctggagtgga cccaatggtc 301 tccttccccg tggccctcag ctgtcactgt ggaccctgcc gcctcagcag cactgactgc 361 gggggtccca gaacccaacc cttggcctgt gaccaccccc cgctcccaga catcctcttc 421 ctctaa