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Y-chromosome-bearing sperm

Novel single-chain fragment variable antibody targeting plasma membrane epitopes on porcine Y-chromosome-bearing sperm



The aim of this study was to construct and produce a single-chain fragment variable (scFv) antibody targeting the plasma membrane epitope on porcine Y-chromosome-bearing sperm (Y-sperm). The hybridoma cloned from 4D1-E8 exhibited the highest specificity, and Y-sperm was used to generate the scFv gene. The expected combination of 450 bp (VH) and 300 bp (VL) resulted in a 795 bp scFv gene. The scFv gene was inserted into the pET22b expression vector and expressed in E. coli BL21 (DE3). The resulting H4:L4 clone produced a highly specific scFv antibody to Y-sperm. 

The reactivity of the H4:L4 scFv antibody to porcine Y-sperm was confirmed via ELISA and flow cytometry. The H4:L4 scFv antibody exhibited low cross-reactivity with X-sperm (4.14%). The soluble H4:L4 scFv antibody exhibited significantly less cross-reactivity with X-sperm than did the 4D1-E8 mAb (4.14% vs. 15.5%). However, H4:L4 scFv and the 4D1-E8 mAbs had high cross-reactivity with other conventional livestock semen. 

The scFv antibodies and mAbs were detected on the Y-sperm surface via immunofluorescence, and the fluorescence intensities were particularly strong on the plasma membranes of Y-sperm. In this study, the production of a scFv antibody against porcine Y-sperm was successful and represents a novel achievement. This scFv antibody had a high affinity for porcine Y-sperm. The soluble scFv antibody and mAb can be used to sort Y- and X-sperm in treatments involving porcine semen bearing X or Y chromosomes.

The successful application of sexed semen technology in livestock production has led to the third revolution of reproductive technology after artificial insemination and embryo transfer. In recent decades, several unsuccessful studies and many inoperative patents for the separation of sperm bearing X and Y chromosomes have emerged. Physical parameters, including density, surface charge, swimming velocity, and sex-specific antigens, have been employed in numerous attempts to differentiate between X-chromosome-bearing sperm (X-sperm) and Y-chromosome-bearing sperm (Y-sperm). 

Currently, there is one quantitative and reasonably accurate method for sexing mammalian sperm that involves individual separation and discrimination of X-sperm and Y-sperm via flow cytometry. In numerous mammalian species, flow cytometry has been implemented to separate sperm, and it has advanced to a stage that permits commercial applications. There are numerous deficiencies in this method, including its high cost, damage to sperm cells, decreased conception rate after artificial insemination, and low sperm dose per straw. 

Consequently, identifying a sperm sorting method that is simple, inexpensive, effective, and less damaging is imperative. In addition, sex sorting via flow cytometry was successful for the bovine production industry. The use of sex sorting technology in pig production systems offers many similar advantages. However, several factors currently limit the implementation of sexing technology in pigs. The anatomical and physiological features inherent to the female pig, together with the relatively low sperm output of the flow cytometer, are the main limitations to the widespread use of this technology in pig production systems. 

Therefore, it is extremely important to develop immunological methods that are effective, cost-effective, convenient, and cause less damage in the process of sorting sperm. Immunological sexing, an alternative semen sexing method, has been successful in bulls, but there have been few studies on this topic in pigs.

Initially, the investigation of immunological sexing requires the initial development of antibodies that are specific to porcine Y-sperm. The surface antigens and specific proteins of X- and Y-sperm differ from each other. Therefore, the ability to evaluate specific antibodies is widely recognized. Engineered antibodies have been generated through the application of recombinant DNA technology. The currently employed practices of animal immunization and hybridoma development can be replaced by a bacterial system that is capable of synthesizing and expressing virtually an infinite quantity of antibodies to nearly any antigen. 

Recombinant antibodies (rAbs) are generated using recombinant DNA technologies, and they can offer several advantages over polyclonal and monoclonal antibodies. The best-known antibody fragments are the antigen-binding fragment (Fab) and the even smaller single-chain variable fragment (scFv). The scFv format consists of the variable region of the heavy chain (VH) joined to the variable region of the light chain (VL) with a peptide linker. They are low-cost and easily produced. Additionally, recombinant antibody affinities can be genetically modified to facilitate their purification or immobilization for downstream applications. 

Producing a scFv antibody for porcine Y-sperm will be both intriguing and beneficial. Previous studies have successfully produced ScFv antibodies specifically for sperm. Thaworn et al. (2022) created a scFv antibody specific to Y-sperm in bovines, and this antibody was successfully applied to sex sorting of bovine semen. In addition, Samuel and Naz (2008) obtained anti-sperm scFv antibodies with defined antigen specificity in humans. Additionally, Soares and Barbosa (2008), for the production of a soluble monoclonal scFv, screened clones from Simental sperm cells via flow cytometry, and those that specifically bound to 40–60% of the cells were selected. 

However, scFv antibody production has not been investigated for porcine Y-sperm. The study of the production of specific scFv antibodies that target Y-sperm is of great interest for application in the pig production industry. A significant amount of interest has been given to the synthesis of specific scFv antibodies against Y-sperm for use in the pig production industry.

Therefore, the present study focused on producing hybridoma cells that produce mAbs and used them to construct a scFv antibody against male-specific sites on a plasma membrane epitope in porcine Y-sperm. Additionally, the effectiveness and specificity of an antigen-binding site in mAbs and scFv antibodies in capturing the plasma membrane of Y-sperm were validated.

Y-chromosome, genetic inheritance, male lineage, paternal ancestry, Y-DNA haplogroups, non-recombining region, genetic markers, STR analysis, SNP mutations, male-specific DNA, human evolution, Y-chromosome sequencing, paternal genealogy, chromosomal aberrations, forensic genetics, ancient DNA studies, molecular anthropology, male infertility genetics, sex determination, population genetics

#Ychromosome, #GeneticInheritance, #MaleLineage, #PaternalAncestry, #YDNA, #Haplogroups, #GeneticMarkers, #SNPs, #STRanalysis, #HumanEvolution, #MolecularAnthropology, #AncientDNA, #PopulationGenetics, #MaleInfertility, #ForensicGenetics, #SexDetermination, #ChromosomeResearch, #GenomicStudies, #GenealogyResearch, #GeneticsResearch

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