Negative biomarker based male fertility evaluation: Sperm phenotypes associated with molecular-level anomalies

Abstract

Biomarker-based sperm analysis elevates the treatment of human infertility and ameliorates reproductive performance in livestock. The negative biomarker-based approach focuses on proteins and ligands unique to defective spermatozoa, regardless of their morphological phenotype, lending itself to analysis by flow cytometry (FC). A prime example is the spermatid specific thioredoxin SPTRX3/TXNDC8, retained in the nuclear vacuoles and superfluous cytoplasm of defective human spermatozoa. Infertile couples with high semen SPTRX3 are less likely to conceive by assisted reproductive therapies (ART) and more prone to recurrent miscarriage while low SPTRX3 has been associated with multiple ART births. Ubiquitin, a small, proteolysis-promoting covalent posttranslational protein modifier is found on the surface of defective posttesticular spermatozoa and in the damaged protein aggregates, the aggresomes of spermiogenic origin. Semen ubiquitin content correlates negatively with fertility and conventional semen parameters, and with sperm binding of lectins LCA (Lens culinaris agglutinin; reveals altered sperm surface) and PNA (Arachis hypogaea/peanut agglutinin; reveals acrosomal malformation or damage). The Postacrosomal Sheath WWI Domain Binding Protein (PAWP), implicated in oocyte activation during fertilization, is ectopic or absent from defective human and animal spermatozoa. Consequently, FC-parameters of PAWP correlate with ART outcomes in infertile couples and with fertility in bulls. Assays based on the above biomarkers have been combined into multiplex FC semen screening protocols, and the surface expression of lectins and ubiquitin has been utilized to develop nanoparticle-based bull semen purification method validated by field artificial insemination trials. These advances go hand-in-hand with the innovation of FC-technology and genomics/proteomics-based biomarker discovery.

Figure 1

Fluorescent labeling of negative sperm quality biomarkers in human and animal spermatozoa. (a) Testis-specific thioredoxin SPTRX3 (red) is retained in the superfluous cytoplasm of defective human spermatozoa. (b) Ubiquitin (red) coats the surface of a defective bull spermatozoon with its flagellum coiled around the head but is undetectable in morphologically normal spermatozoa. (c) Aggresomes (red) are stress-induced aggregates of ubiquitinated proteins, here detected by ProteoStat kit in the mitochondrial sheath and head of a defective boar spermatozoon. (d) Sperm head postacrosomal protein PAWP (red) is detected at varied intensities in bull spermatozoa. (e) Lectin LCA (green) binds exclusively to the acrosomes of phenotypically normal bull spermatozoa but to the whole head and tail surface in the defective ones (segmental aplasia of the mitochondrial sheath is shown). (f) Lectin PNA binds to damaged/ruffled acrosomes, but not the intact ones in live bull spermatozoa. DNA in panel (a) was counterstained blue with DAPI. Epifluorescence micrographs are superimposed over parfocal images taken with DIC optics. PAWP: postacrosomal Sheath WWI Domain Binding Protein; LCA: Lens culinaris agglutinin; PNA: peanut agglutinin; DIC: differential interference contrast.