Skip to main content

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

International Conference on Genetics and Genomics of Diseases

Visit: genetics-conferences.healthcarek.com

Award Nomination: genetics-conferences.healthcarek.com/award-nomination/?ecategory=Awards&rcategory=Awardee

Award registration: genetics-conferences.healthcarek.com/award-registration/

For Enquiries: contact@healthcarek.com

Get Connected Here
---------------------------------
---------------------------------
in.pinterest.com/Dorita0211
twitter.com/Dorita_02_11_
facebook.com/profile.php?id=61555903296992
instagram.com/p/C4ukfcOsK36
genetics-awards.blogspot.com/
youtube.com/@GeneticsHealthcare

    Comments

    Post a Comment

    Popular posts from this blog

    Genetics role in ovarian cancer

    The Medical Minute: Genetics play big role in ovarian cancer In 2024, about 19,680 women in the United States will receive a new diagnosis of ovarian cancer and 12,740 women will die from the disease, said Dr. Shaina Bruce , a gynecologic oncologist at Penn State Cancer Institute . The median age of all patients who develop ovarian cancer is 63. Historically, women at increased risk for ovarian cancer are recommended to have their fallopian tubes and ovaries removed when they have completed having children. Taking that step to protect themselves comes at a heavy price ― surgical menopause. But Bruce said medical science is catching up with ovarian cancer. Studies could lead to new methods for preventative care and the surgery needed to lower risk may be easier than it once was. Below, during Gynecologic Cancer Awareness Month, Bruce discusses the disease and why acting to reduce your risk is worth it. What’s the connection between heredity and ovarian cancer? About 25% of all cases of ...
    Post a Comment
    Read more

    Multifactorial Genetic Conditions

    Multifactorial Genetic Conditions Multifactorial genetic conditions are disorders caused by the combined effects of multiple genes and environmental factors , rather than a single gene mutation . These conditions do not follow classic Mendelian inheritance patterns and instead result from complex gene–environment interactions . Factors such as lifestyle, nutrition, infections, stress, and exposure to toxins can significantly influence disease onset and severity in genetically susceptible individuals. Common examples include diabetes, cardiovascular diseases , neural tube defects, asthma, and many neuropsychiatric disorders. Understanding multifactorial inheritance is essential for risk prediction, preventive medicine, and personalized healthcare strategies. Multifactorial inheritance, polygenic traits, gene–environment interaction, complex diseases, genetic susceptibility, environmental risk factors, non-Mendelian inheritance, disease predisposition, polygenic risk score, precision ...
    Post a Comment
    Read more

    X chromosome

    Gene on the X chromosome may help explain high multiple sclerosis rates in women Brain inflammation may be fueled by a gene on the X chromosome, a new study in mice suggests. And in female mice, who carry two X chromosomes, a diabetes drug called metformin may work to counteract that inflammation. If these findings bear out in later studies, they could help to unravel the long-standing mystery of why women, who have two copies of this inflammation-driving gene, are more prone to certain autoimmune diseases, particularly after menopause. A disparity between the sexes Our bodies are patrolled by immune cells that provide protection against bacteria and viruses, but sometimes, these defenses turn on us. In the autoimmune disorder multiple sclerosis (MS), for instance, the immune system attacks myelin, the fatty insulation surrounding the nerve fibers in the brain and spinal cord. This leads to symptoms such as muscle weakness and difficulty walking, as well issues with memory and thinking...
    Post a Comment
    Read more