Application of a New Convenience Gender Sorting Method for Mouse Spermatozoa to Mouse Reproductive Engineering Technology

Abstract

ABSTRACT. In this study, we attempted to apply new convenience gender sorting methods using sex-determining region Y (SRY) gene expression on Y spermatozoa to mice. Mouse spermatozoa labeled with Cy3-SRY antibody conjugate were used for intracytoplasmic sperm injection (ICSI). In addition, spermatozoa conjugated with SRY antibody were conjugated with magnetic beads (Mag) and were pulled to the bottom of the medium. The supernatant of the medium was used for in vitro fertilization (IVF). The rate of males reproduced by ICSI using the spermatozoa conjugated with Cy3-SRY antibody was 86.1%. The female proportion reproduced by IVF using the spermatozoa separated in the supernatant after Mag-SRY antibody conjugation was 67.3%. These gender sorting methods are effective for the reproduction of transgenic mice. KEY WORDS: gender sorting, in vitro fertilization, Y spermatozoa.doi: 10.1292/jvms.12-0303; J. Vet. Med. Sci. 75(2): 231-235, 2013 The goal of this research was to establish a new convenience gender sorting method for mammals, which has been difficult in the past. As a first step, a mouse reproductive engineering technology was established. The establishment of gender sorting technology is essential for the reproduction of laboratory animals [7], livestock [1,3,6] and humans [2,5,8,15]. The reproduction of mice and rats by reproductive engineering technology included intracytoplasmic sperm injection (ICSI), in vitro fertilization (IVF) and embryo transfer (ET), which are important technologies in laboratory animal facilities. Therefore, we attempted gender sorting targeted at mice with an established reproductive technology. There is high demand for females for the production of mice. For example, the reproduction of mice carrying several mutant genes, such as IRS-1 deficient Gck −/+ mice [12] and PPARγdeficient ob/ob mice [9], using reproductive engineering technology requires many females in the production process, although a male is required in the final stage. When no accidental bias occurs, the gender ratio after ET and Cesarean section is usually 50:50 (female:male). Modi et al. (2005) reported that SRY transcription occurs in spermatozoa in humans [10]. From their study, we assumed that the same phenomenon occurs in mouse spermatozoa. If this was true, then the sex-determining region Y (SRY) protein may also be produced by spermatozoa. Therefore, we examined the previous report indicating that SRY transcription occurs in spermatozoa in humans [10] and contrived a new technology for gender sorting using fluorescence or magnetic beads via SRY antibody, using mice as an example of mammals.Because the SRY gene consists only of an exon, SRY gene expression was confirmed by two polymerase chain reaction (PCR) experiments using primers designed with a specific sequence of intra-exons to detect cDNA or a sequence ranging from the 5'-genomic region to the exon to detect genomic DNA (Fig. 1). To generate positive control vectors, RNA and genomic DNA were extracted from the sperm and testes of C57BL/6JJcl mice at...

​Spermatozoa Sorting Techniques for the Sex Pre-selection: A Review

“…In a study conducted in mice, researchers have conjugated the Cy3-SRY antibody-containing a magnetic bead to pull down the Y-spermatozoa. Higher purity of Y-spermatozoa was obtained than X-spermatozoa (Hashimotoa et al, 2013). Magnetic nanoparticle-based separation based on the interaction between the magnetic nanoparticle and zeta potential of spermatozoa employed in donkeys resulted in 90% purity of X-spermatozoa (Domínguez et al, 2018).…”

Antibody-Conjugated Magnetic Beads for Sperm Sexing Using a Multi-Wall Carbon Nanotube Microfluidic Device

“…In the past 10 years, lab on a chip (LOC) has supported sperm analysis and sorting discussed by Samuel et al [ 10 ]. By designing of micropatterning of microfluidic channel, LOC integrated with sensors and actuators are employed such as fluorescence-activated cell sorting (FACS) [ 11 , 12 ], dielectrophoretic sorting [ 13 ], electrophoretic sorting [ 14 , 15 , 16 ], magnetic-activated sperm cell sorting [ 17 , 18 , 19 ], sperm sorting to utilize the difference in motility or nonmotility [ 20 , 21 , 22 , 23 , 24 , 25 ], immunological markers [ 26 , 27 ], electrokinetic isolation [ 28 ], inertial separation [ 29 ], and controlled pressure sorting [ 30 ]. Another simple technique which improved to acquire higher effective by optimizing microfluidic chip parameters based on electrophoresis was proposed with successful sorting up to an accuracy of 70% [ 16 ].…”

Antibody-Conjugated Magnetic Beads For Sperm Sexing By A Multi-Wall Carbon Nanotube Microfluidic Device

“…Successful applications of micro uidic systems for sperm sorting and analysis have been discussed by Samuel et al [12] . In addition, sperm separation methods such as uorescence-activated cell sorting (FACS) [13,14] , dielectrophoretic sorting [15] , electrophoretic sorting [16][17][18] , magnetic activated sperm cell sorting [19][20][21] , sperm sorting to utilize the difference in motility or nonmotility [22][23][24][25][26][27] , immunological markers [28,29] , electrokinetic isolation [30] , inertial separation [31] , and controlled pressure sorting [32] are used to isolate homogeneous and concentrated cell populations from heterogeneous cell mixtures. Each sorting method has its own advantages and disadvantages.…”