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Antibody for Determining Sex of Sperm, and Use Thereof

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Antibody for Determining Sex of Sperm, and Use Thereof (19) TZZ¥¥ ¥ _T (11) EP 3 323 828 A1 (12) EUROPEAN PATENT APPLICATION published in accordance with Art. 153(4) EPC (43) Date of publication: (51) Int Cl.: 23.05.2018 Bulletin 2018/21 C07K 14/705 (2006.01) C07K 16/28 (2006.01) G01N 33/53 (2006.01) A01K 67/02 (2006.01) (21) Application number: 16824713.8 (86) International application number: (22) Date of filing: 13.07.2016 PCT/KR2016/007582 (87) International publication number: WO 2017/010796 (19.01.2017 Gazette 2017/03) (84) Designated Contracting States: (71) Applicants: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB • Kim, Dong Ku GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO Seoul 01603 (KR) PL PT RO RS SE SI SK SM TR • Nuriscience Co., Ltd. Designated Extension States: Seoul 05053 (KR) BA ME Designated Validation States: (72) Inventor: Kim, Dong Ku MA MD Seoul 01603 (KR) (30) Priority: 13.07.2015 KR 20150099080 (74) Representative: Brevalex 12.07.2016 KR 20160088170 56, Boulevard de l’Embouchure B.P. 27519 31075 Toulouse Cedex 2 (FR) (54) ANTIBODY FOR DETERMINING SEX OF SPERM, AND USE THEREOF (57) The present invention relates to an antibody for an agglutination reaction of Y-chromosome bearing determining the sex of a sperm, and a use thereof and, sperm through the treatment of the antibody, and thus more particularly, to a composition for determining the customized animals having a particular sex can be sex of a sperm, a method for determining sex, and a mass-produced. In addition, livestock having a desired method for producing an animal having a particular sex, sex is selectively produced, and thus the present inven- the composition and the methods using the antibody. It tion is expected to contribute to planned breeding, an has been confirmed, in the present invention, that an improvement in breeding, and an improvement in man- X-chromosome bearing sperm and a Y-chromosome agement efficiency. bearing sperm can be easily distinguished by inducing EP 3 323 828 A1 Printed by Jouve, 75001 PARIS (FR) EP 3 323 828 A1 Description TECHNICAL FIELD 5 [0001] The present disclosure relates to an antibody for sexing sperms and use thereof, and more particularly, to a composition for and a method of sexing sperms by using the antibody, and a method of producing an animal of a particular sex. BACKGROUND ART 10 [0002] Sperm sexing biotechnology is a technology for producing offspring of a desired sex by separating only sperms of the desired sex and inducing fertilization and pregnancy. This technology is very important in production efficiency, management, and commercial aspects, and the need for technology development is growing. [0003] In particular, in a variety of in vivo or in vitro protocols such as fertilized egg production and artificial insemination, 15 selection of a desired sex may have great economic benefits. For example, in the case of cows, female calves capable of producing milk may be selectively produced, and in the case of beef cattle, male calves capable of efficiently producing more meat than female calves because of a high growth rate and a high weight gain may be selectively produced, or high-yielding female cattle for the purpose of breeding improvement for the production of high-quality beef cattle may be selectively mass-produced, contributing to efficient management in the livestock farmers and economic benefit there- 20 from. [0004] Meanwhile, with regard to sex determination of livestock, the sex of offspring during pregnancy is determined by a combination of two chromosomes constituting the sex chromosome pair, and consequently, determined by the chromosome of a sperm, that is, the sex of the sperm. [0005] Specifically, sperms present in semen of livestock are divided into female (X chromosome) sperms and male 25 (Y chromosome) sperms, and diploid spermatocytes in the testicular tissue differentiate into mature haploid spermatozoa. 50% of spermatozoa bearing X chromosome and 50% of spermatozoa bearing Y chromosome exist in the semen. Therefore, the sex of offspring is determined by the pair of sex chromosomes (XX or XY) formed by fertilization of an egg (X chromosome) by a sperm bearing any one of X chromosome and Y chromosome in the semen. Therefore, a nature female-to-male sex ratio is generally 50 : 50 of female offspring: male offspring. 30 [0006] Practically, the importance and necessity of a sex-controllable technology has been recognized for a long time in many fields including livestock production, and a variety of techniques for sorting X sperm bearing X chromosome and Y sperm bearing Y chromosome by a physical method or a morphological method has been attempted. [0007] In 1982, since scientists at the US Department of Agriculture, the University of Colorado, and the University of Cambridge, UK reported that sexing of sperms may be conducted based on a difference in a DNA content according to 35 lengths of X and Y chromosomes of sperms, various researches have been conducted up to date. For example, methods of sorting sperms based on a migration rate according to sperm motility are disclosed, in which centrifugation is performed or sperms are passed through a bead layer, columns made of various materials, etc., based on the size, weight and density of sperms, (Korean Patent Publication NO. 10-2009-0024034), but there is a problem that it is difficult to obtain sperms of a desired sex with high purity only by these methods. 40 [0008] Further, a technology of Sexing Technologies (transfer of rights from XY LLC) for sexing sperms by a length difference between X and Y chromosomes is now commonly used, in which chromosomes of sperms are stained with a fluorescent stain for a short time, and then scattered light generated by a laser attached to a cell sorter is converted into electric signals to determine the sex of sperms, or sperms are passed through a high pressure separator to separate desired sperms. 45 [0009] These mechanical sorting processes cause loss of many sperms without sorting, and significantly affect motility and a survival rate of sperms, which are crucial for fertilization with oocytes, due to the long-term sorting process and stress by laser and electric stimulation. Further, since sperms are sorted by using the device, they are sold at about 4 times higher price than non-sorted semen, and the number of sperms stored in commercial frozen semen straws is about 5-10 times less than the number of non-sorted sperms, which is a major cause of low pregnancy rates. 50 [0010] Further, since sperm sorting by the above method requires expensive equipment and specialized personnel, and additional facilities and manpower are absolutely needed for mass production, the related industries are facing many difficulties due to the limitations in the sexing of sperms by the device. Furthermore, the most important concern is that since the fluorescent material used for sperm sorting is nonspecifically bound to all chromosomes and fertilization occurs without being removed before fertilization with oocyte, there are many doubts about the genetic safety of offspring. In 55 Europe, where animal welfare is the top priority, discussions are underway to ban the sperm sorting method. [0011] Currently, there is an urgent need to develop a new technique to overcome the problems of the above-described sperm sorting method using the device. Many scientists are studying in order to find out a biomarker capable of distin- guishing between X sperm and Y sperm. 2 EP 3 323 828 A1 DETAILED DESCRIPTION OF THE INVENTION TECHNICAL PROBLEM 5 [0012] In order to solve the above problems, the present inventors developed an antibody specifically binding to Y chromosome sperms, and they found that desired X chromosome sperms or Y chromosome sperms may be sorted by using the antibody, leading to sexing of sperms, thereby completing the present disclosure. [0013] Accordingly, an object of the present disclosure is to provide an antibody including a heavy chain variable region including any one or more selected from the group consisting of HCDR1 including an amino acid sequence 10 represented by SEO ID NO: 1, HCDR2 including an amino acid sequence represented by SEO ID NO: 2, and HCDR3 including an amino acid sequence represented by SEQ ID NO: 3; and a light chain variable region including any one or more selected from the group consisting of LCDR1 including an amino acid sequence represented by SEQ ID NO: 4, LCDR2 including an amino acid sequence represented by SEQ ID NO: 5, and LCDR3 including an amino acid sequence represented by SEQ ID NO: 6. 15 [0014] Further, another object of the present disclosure is to provide a composition for sexing sperms including the antibody as an active ingredient and/or a related product including a kit and/or a method of applying the same. [0015] Further, still another object of the present disclosure is to provide an expression vector for producing the antibody, including isolated nucleotides encoding amino acid sequences including SEQ ID NO: 7 or SEQ ID NO: 8, a host cell transformed with the expression vector in vitro, and a method of producing the antibody by using the host cell. 20 [0016] Further, still another object of the present disclosure is to provide a method of sexing sperms of a mammal, the method including a) collecting semen from a male subject; b) treating the semen with the antibody; and c) sorting sperms specifically bound to the antibody and non-bound sperms in the semen. [0017] Further, still another object of the present disclosure is to provide a method of producing a mammal of a particular sex, the method including a) collecting semen from a male subject; b) sexing sperms by treating the semen with the 25 antibody; c) performing fertilization by using the sexed sperms; and d) producing offspring of the particular sex.
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