Olumide Olaseni Adenmosun, M.S., M.B.A., James KumiDiaka, D.V.M., Ph.D., Waseem Asghar, Ph.D.
Florida Atlantic University
In this review, we discuss the potential of the mouse/human H-Y 2.0 antigen as the ideal molecular biomarker of choice for sperm sexing protocols. Methods of separating X-chromosome and Y-chromosome bearing sperm have been extensively discussed in literature focused on assisted conception techniques for use in human clinical applications and in veterinary medicine for improved animal husbandry. The most successful of these methods of sperm sexing is flow-cytometric sorting which has been met with limitations and clinical concerns ranging from mutagenic predispositions to significant decrease in yield (sperm concentration) and reduced fertility potentials of sorted sperms. A revision of the amino acid sequence of the H-Y antigen which has the lowest homolog frequency in the SMCX gene is being evaluated to create efficient monoclonal antibodies for a proper immuno-sort and enrichment of the X and Y sperm for clinical and veterinary applications.
The H-Y antigen is a male specific minor histocompatibility antigen present on the SMCY gene of the Y-chromosome. The antigen is known to elicit rejection of male organs or grafts in female recipients receiving transplant. Even when there seem to be a match at the human major histocompatibility locus between a transplant recipient and a donor, a rejection can still be induced despite the close relatedness of the H-Y antigen and its homolog on the SMCX gene of the X-chromosome (1). Apart from the development of graft-versus-host-disease (GVHD) in female recipients, as a result of the male-specificity of the H-Y antigen, the protein has also been evaluated as a potential marker for sorting X and Y-chromosome bearing sperm which are utilized in assisted conception procedures for the prevention of genetic disorders or for gender balancing (2).
MOLECULAR CHARACTERISTICS OF SMCY-DERIVED H-Y ANTIGEN
The H-Y antigen is specifically present on the SMCY gene of the Y-chromosome at the locus Yq11.223. The H-Y antigen also has aliases called HYA, SMCY, JARID1D, and KDM5D (lysine demethylase 5D) among others. The H-Y protein is expressed in most male tissues including the prostate and testes. It has also been found on the sperm surface membrane as a major antigen that has the potential of delineating sperm cells that carry the X or the Y-chromosome (3). SMCY encodes a zinc-finger domain protein and its main molecular function has been associated with DNA binding and transcriptional regulation. Microdeletions or mutations in the SMCY gene can also result in spermatogenic dysfunction (4). Studies have shown that the protein encoded by the gene is also expressed in other body tissues including the small intestine, urinary bladder, gall bladder, stomach, duodenum, brain and the bone marrow, however it is absent in endometrial and ovarian tissues (5). The broad sequence of the SMCY gene has about 28 exons where the H-Y antigen has been shown to have several epitopes (4). Therefore, the size of the H-Y antigen varies considerably from an average of 9 to 11 amino acid residues which have been derived from the evolutionarily conserved structure of the SMCY gene in humans and mice (1, 4).
H-Y HOMOLOG ON SMCX GENE
The SMCX gene on the other hand is present on the X-chromosome at the locus Xp11.22. It is also associated with the following aliases: MRXJ, MRX13, JARID1C, and KDM5C (lysine demethylase 5C) among others. The SMCX gene is regarded as a member of the SMCY homolog family, and its primary molecular function also involves the regulation of DNA transcription and chromatin remodeling. Mutations in the gene have also been reported to be associated with X-linked cognitive disability (6). The protein encoded by SMCX is broadly expressed in male and female tissues, including the endometrium, ovary, prostate, and testis, among others. Beyond an 84% amino-acid similarity between the SMCY/SMCX gene in mouse, another close homolog of both genes has been identified as the human retinoblastoma binding protein 2 (RBP2) (4). A study also associated the müllerian-inhibiting substance (MIS) as a direct homolog of the H-Y protein which cross reacts with an H-Y antiserum (7). The SMCX sequence in mice from a study, as populated from a partial clone that was completed by RT (Reverse Transcription) and RACE (Rapid Amplification of cDNA Ends) PCR (Polymerase Chain Reaction) techniques, showed a total of 5673bp while the SMCY sequence had 5316bp with exonic regions showing a total of 4656bp for SMCX and 4647bp for SMCY, respectively. An analysis of the alignment of both SMCX/SMCY for two possible H-Y epitopes in the Y chromosomal sequences of both human and mouse genes showed an average match of 90.9% and 75.24% H-Y sequence homology in humans and mice respectively (4).
PREVIOUS ATTEMPTS AT SPERM SEXING WITH H-Y
Considering the significant homologous sequences for H-Y antigenic epitopes derived from the SMCX and SMCY genes (8), some research groups are still trying to devise a reasonable scientific pathway to creating the most efficient monoclonal antibody against the H-Y antigen to help sort or enrich Y-chromosome bearing sperm cells for animal breeding purposes or for clinical application in humans (for assisted conception procedures).
A study conducted in 1998 by Sills et al. (3), using an immuno-magnetic bead separation method that was further confirmed by fluorescent in situ hybridization, described current immunologic sperm sexing techniques that solely relied on the H-Y antigen as unlikely to help achieve sex selection. The result of the study specifically revealed that 54.1% of sorted sperm cells contained Y-chromosomes while 49% of the rest of the magnetically non-reactive group were detected by FISH (Fluorescent in situ hybridization) to have Y-chromosomes despite not being reactive to the anti-H-Y immuno-beads. The summary of that sperm sexing trial showed that H-Y antigen expression on sperm surface membranes was non-random and nonspecific. However, there was still a slightly higher frequency of the Y-chromosome in the Y-sorts but the expression of the H-Y antigen or its possible homolog on the X-chromosome bearing sperm was also considerable.
Several other studies have detailed the failure of the H-Y antigen as an inefficient sperm sexing marker, but an older study by Ali et al. (10) once reported a successful enrichment of bovine X and Y-chromosome bearing sperm with H-Y monoclonal antibodies, which were further counted with a fluorescent microscope and a fluorescent-activated cell sorter (FACS). The results of three H-Y immuno-sorted bovine sperm yielded ratios of 76:24, 88:12, and 77:23 Y to X-chromosome bearing spermatozoa, respectively; and a negative sort ratio (nonfluorescent sperm) of 26:74, 35:65, and 23:73, respectively, against a 50:50 ratio for non-sorted control samples.
CONVENTIONAL FLOW-CYTOMETRIC SPERM SORTING: SUCCESS AND CHALLENGES
Besides the H-Y immuno-sorting method, the most successful sperm sexing technique that has yielded the most enrichment results (90% X-sort and about 70% Y-sort) has been the flow-cytometric method (11). It requires that the sperm cells be initially stained with a DNA-binding dye (Hoechst 33342) which will have to penetrate the sperm membrane to stain the dense chromatin within the sperm head. The principle behind flow cytometric sperm sorting rests on the 2.8% difference in the DNA content between the X and Y-chromosome bearing sperm. The X-chromosome bearing sperm has a slightly higher DNA content and thus it stains more intensely with the fluorescent dye which can be detected by the flow cytometer. It is also known that the X-chromosome bearing sperm, due to its slightly denser DNA content and additionally expressed total surface protein content like sialic acid (12), has a higher net negative charge which can also be preferentially sorted by the flow cytometer. However, with all the prospects of the flow cytometric sorting method, it has also been mired with significant clinical concerns such as safety of the technique. The use of potentially mutagenic agents utilized in the sorting methodology (which are the DNA-binding dye and the ultraviolet/laser detectors in the flow-cytometric system) may predispose the sperm cells to non-physiologic conditions and iatrogenic damage (13). A clinical trial update on the use of MicroSort, which was solely dependent on the flow-cytometric sorting method showed that sperm yield for IUI (Intra-uterine insemination) from a post-sort is considerably lower than generally reported for IUI procedure with unsorted sperm (12).
SPERM SEXING BY ELECTROPHORETIC MOBILITIES
Some very early studies (12) have also attempted to sort the X-chromosome bearing sperm cell from the Y-chromosome bearing sperm cell by their respective electrophoretic mobilities when subjected to an electric field and a neutral lateral flow medium to measure their net velocities or how fast they swim-through in a forward progressive direction. The slight weight difference in the chromatin content (2.8% more) of the X-chromosome bearing sperm cell – being heavier than the Y-chromosome bearing sperm cell seem to make the X-sperm lag slightly behind the Y-sperm in a neutral field (16). However, the phenomenon seems reversed when the electrophoretic mobilities of X-chromosome bearing sperm cells were observed in an electric field, the higher net negative charge on the X-sperm due to a high sialic acid content enables them to have a higher electrophoretic mobility towards the anodic region than the Y-sperm (12). Another conflicting study however refuted the earlier notion that X-sperm were more negatively charged but rather asserted that Y-sperm have a more net negative charge when separated electrophoretically (16). Therefore the net weight and the electrophoretic mobility potentials seem to be unreliable for adoption as efficient sperm sexing methods.
With all the limitations of the current sperm sexing methods, it may be important to note that a viable selectable marker on the sperm surface membrane with some bias for the Y-chromosome bearing sperm remains the H-Y antigen. But with the mediocre results and failures at achieving a clinically relevant and statistically significant sort result, it may be important to review the composition of the antigenic epitopes that are being utilized in the creation of the H-Y antisera or monoclonal antibodies currently in use for sperm sexing (9).
Some studies have already attempted an extensive rummage through a thicket of possible differentially expressed proteins between the X and Y spermatozoa that may lead to variations in phenotypic characteristics. A study by Chen et al. (14) utilizing the spermatozoa of bulls subjected to protein analytics using methods like 2-dimensional electrophoresis (2-DE) and mass spectrometry with other proteomic technologies like western blot analysis gave rise to over 42 significant protein spots that are known to be differentially expressed between the X and Y sperm. Newly identified proteins to delineate X and Y sperm, from these analyses were associated with functional categories like energy metabolism, cellular defense and stress, cytoskeleton and inhibitor of serine proteases. However, the possible limitations to the adoption of some of these newly identified proteins like CAPZB and UQCRC1 would depend on the site of expression on/in the sperm cell and the likelihood that these proteins are not shared between the X and Y sperm because of intercellular bridges that exists among sperm of the same origin.
RAISING mabs FOR H-Y 2.0
The ideal H-Y antigen to use as a selectable marker for sperm sexing would be dependent on the point of difference in the SMCY and SMCX homologs at the variation in their H-Y amino acid sequence. Since the flaws of the sperm-immuno-sort technique using the H-Y antigen have been extensively discussed in numerous studies, a re-adoption of a well conserved epitope with the lowest sequence alignment to an SMCX-derived H-Y homolog as represented in Figure 1 would be the appropriate protein to use for the creation of an efficient monoclonal antibody (mab), using a hybridoma development protocol. Further bioinformatics analytics can be performed to re-analyze and re-confirm the alignment frequencies of the H-Y antigen homologs on the SMCX/SMCY genes in order to arrive at the ideal H-Y 2.0 antigen. A detailed discussion on the creation of the ideal H-Y 2.0 mab may be the basis of an explorative proteomics research where the best antigenic-epitope candidate would have a lower amino-acid sequence similarity to possible SMCX-derived H-Y homologs.
While other sperm sexing strategies and methods may be continually tested or explored, the H-Y antigen remains a promising selectable biomarker whose expression on the sperm surface membrane, although non-specific, has a higher frequency of expression in the Y-chromosome bearing sperm. Therefore, H-Y antigen has the potential of becoming the ideal sperm sexing molecular target if its immunogenic sequence is further revised.
Along with the other current assisted reproductive technologies being bio-ethically evaluated, such as preimplantation genetic screening or gender selection and balancing, it is our hope that if such scientific explorations lead to the discovery of an ideal antigenic epitope to effectively sort the X-sperm from the Y-sperm, its use will be regulated and limited to clinical cases of familial X or Y-linked genetic disorder inheritance. And its use in the veterinary community may be widely adopted for purposes of improved animal husbandry and selection of healthy animal breeds.
1. Wang W, Meadows LR, den Haan JMM, Sherman NE, Chen Y, Blokland E, et al. Human H-Y: a male-specific histocompatibility antigen derived from the SMCY protein. Science 1995;269(5230):1588-90.
2. Yadav SK, Gangwar DK, Singh J, Tikadar CK, Khanna VV, Saini S, et al. An immunological approach of sperm sexing and different methods for identification of X- and Y-chromosome bearing sperm. Vet World 2017;10(5):498-504.
3. Sills ES, Kirman I, Colombero LT, Hariprashad J, Rosenwaks Z Palermo GD. H-Y antigen expression patterns in human X- and Y-chromosome-bearing spermatozoa. Am J Reprod Immunol 1998;40(1):43–7.
4. Agulnik AI, Longepied G, Ty MT, Bishop CE, Mitchell M. Mouse H-Y encoding Smcy gene and its X chromosomal homolog Smcx. Mamm Genome 1999;10(9):926–9.
5. Fagerberg L, Hallstrom BM, Oksvold P, Kampf C, Djureinovic D, Odeberg J, Uhlen M. Analysis of the human tissue-specific expression by genome-wide integration of transcriptomics and antibody-based proteomics. Mol Cell Proteomics 2013;13(2):397–406.
6. Emily B, Benoit L, Katrin Õ, Renee C, John BM, Michael F, Yang S. Mutations in the intellectual disability gene KDM5C reduce protein stability and demethylase activity. Hum Mol Genet 2015;24(10):2861-2872.
7. Müller U. H-Y antigens. Hum Genet 1996;97(6):701–4.
8. Yao C, Wang Z, Zhou Y, Xu W, Li Q, Ma D, Qiao Z. A study of Y chromosome gene mRNA in human ejaculated spermatozoa. Mol Reprod Dev 2010;77(2):158–66.
9. Chigurupati SP, Rangasamy S, Satheshkumar S. Sex Preselection in domestic animals - current status and future prospects. Vet World 2010;3(7):346-8.
10. Ali JI, Eldridge FE, Koo GC, Schanbacher BD. Enrichment of bovine X-and Y-chromosome-bearing sperm with monoclonal H-Y antibody-fluorescence-activated cell sorter. Arch Androl 1990;24(3):235-45.
11. Karabinus DS. Flow cytometric sorting of human sperm: MicroSort® clinical trial update. Theriogenology 2009;71:74–9.
12. Kaneko S, Oshio S, Kobayashi T, Iizuka R, Mohri H. Human X- and Y-bearing sperm differ in cell surface sialic acid content. Biochem Biophys Res Commun 1984;124(3):950–5.
13. Caroppo E. Sperm sorting for selection of healthy sperm: is it safe and useful? Fertil Steril 2013;100(3):695–6.
14. Chen X, Zhu H, Wu C, Han W, Hao H, Zhao X, et al. Identification of differentially expressed proteins between bull X and Y spermatozoa. J Proteomics 2012;77:59–67.
15. Shen Y, Wang Y, Jiang X, Lu L, Wang C, Luo W, et al. Preparation and characterization of a high-affinity monoclonal antibody against human epididymis protein-4. Protein Expr Purif 2018;141:44–51.
16. Koh J, Marcos. The study of spermatozoa and sorting in relation to human reproduction. Microfluid Nanofluid 2015;18(5/6):755-774.
Partial data showing predicted amino acid alignment sequence for human SMCX/SMCY and mouse Smcx/Smcy genes. Human: SMCX and SMCY, accession numbers: U52191 and L25270, respectively; Mouse: Smcx and Smcy, accession numbers: AF127245 and AF127244, respectively. Dots indicate a match with the human SMCX amino acid sequence. H-Y epitopes are underlined in the mouse and human Y chromosomal sequences (human: HLA-B*0702; mouse: H-Y/D[b]). Sequence data extracted from Agulnik et al. (1999) (4).