Biomarker-based human and animal sperm phenotyping: the good, the bad and the ugly†

Abstract

Conventional, brightfield-microscopic semen analysis provides important baseline information about sperm quality of an individual; however, it falls short of identifying subtle subcellular and molecular defects in cohorts of "bad," defective human and animal spermatozoa with seemingly normal phenotypes. To bridge this gap, it is desirable to increase the precision of andrological evaluation in humans and livestock animals by pursuing advanced biomarker-based imaging methods. This review, spiced up with occasional classic movie references but seriously scholastic at the same time, focuses mainly on the biomarkers of altered male germ cell proteostasis resulting in post-testicular carryovers of proteins associated with ubiquitin-proteasome system. Also addressed are sperm redox homeostasis, epididymal sperm maturation, sperm-seminal plasma interactions, and sperm surface glycosylation. Zinc ion homeostasis-associated biomarkers and sperm-borne components, including the elements of neurodegenerative pathways such as Huntington and Alzheimer disease, are discussed. Such spectrum of biomarkers, imaged by highly specific vital fluorescent molecular probes, lectins, and antibodies, reveals both obvious and subtle defects of sperm chromatin, deoxyribonucleic acid, and accessory structures of the sperm head and tail. Introduction of next-generation image-based flow cytometry into research and clinical andrology will soon enable the incorporation of machine and deep learning algorithms with the end point of developing simple, label-free methods for clinical diagnostics and high-throughput phenotyping of spermatozoa in humans and economically important livestock animals.

Keywords: biomarker; fertility; phenomics; proteome; sperm.

Graphical Abstract

Figure 1

Imaging of testicular and epididymal tissues and apical blebs in transgenic mice expressing the enhanced green fluorescent protein (EGFP) insert driven by ubiquitin C (UBC) gene promoter. Epifluorescence images (A–C) are complemented by transmission electron micrographs (D–F). (A) Testicular tissue section showing the accumulation of EGFP in the adluminal compartment of the seminiferous tubules, corresponding to the cytoplasmic lobes of elongating spermatids. Red autofluorescence outlines the interstitial tissue. (B) Accumulation of EGFP protein in the secretory sites of the adluminal compartment of UBC-GFP mouse caput epididymis. Arrows point to longitudinal sections and arrowheads to cross-sections of apical blebs. (C) Intense EGFP fluorescence in the epididymal clear cells (arrowheads) implicated in endocytosis of the soluble and phagocytosis of solid components of epididymal fluid. (D–F) Transmission electron micrographs of mouse caput epididymis showing cross-sections (D) and longitudinal (E) sections of apical blebs, and a longitudinal section of a clear cell (F). See Supplementary Data File S1 for details and additional images.

Figure 2

Imaging of EGFP in the epididymal tissue sections of transgenic rats expressing EGFP construct driven by ubiquitin C (UBC) gene promoter. High intensity of green fluorescence identifies cells with high transcriptional activity of UBC gene and resultant accumulation of EGFP. (A) shows a low-magnification view while [(B) including inset] zooms in on apical blebs. High EGFP intensity in the blebs indicates their ability to concentrate and release (into epididymal lumen) cytosolic proteins that otherwise would not be secreted due to a lack of exocytosis-determining domains. See Supplementary Data File S1 for details and additional images.

Figure 3

Differential lectin labeling patterns in fresh bull spermatozoa. (A) Caragana arborescens agglutinin (CAA) binds to complex glycans and coats the surface of fresh bull spermatozoa with faint red labeling. However, in cells with compromised membrane integrity, indicated by PNA (green) labeling, and those with abnormal morphology, CAA binding is much more prominent. (B) Lens cullinaris agglutinin (LCA) is a mannose binding lectin that is not detected on the surface of all fresh bull spermatozoa but does show binding affinity in cells with certain abnormal morphologies such as the tail defect shown. (C) Phaseolus limensis agglutinin (LBA/lima bean agglutinin) is a GalNAc binding lectin that shows affinity for the postacrosomal sheath region of the sperm head in select sperm cells that also label with PNA (red in this panel), a marker for acrosomal membrane integrity. (D) Ulex europaeus agglutinin (UEA1) is a fucose binding lectin that shows a similar binding affinity to LBA, labeling the postacrosomal sheath region of the sperm head in select cells. (E) Limax flavus agglutinin (LFA) is a sialic acid binding lectin that shows affinity for the tail midpiece in select cells.

Figure 4

Visualization of bull sperm acrosomes (A, B) and spermatid acrosomal granules/caps (C, D) by using lectin PNA (peanut agglutinin; green). Spermatozoa with outward, beaded (A) and inward, indented (B) knobbed acrosomes in a bull carrying a rare, deleterious point mutation of EML5 gene. Imaging of acrosome biogenesis on bull testicular tissue shows acrosomal granules in the early-step round spermatids (C) and acrosomal caps in the elongating spermatids (D). DNA was counterstained with DAPI.

Figure 5

Image gallery of multiplex sperm labeling generated by Cytek Amnis FlowSight imaging flow cytometer. Frozen–thawed bull spermatozoa were labeled with FluoZin-3, AM (zinc ion reporter and capacitation status indicator, green); CellROX Orange (reactive oxygen species probe, orange); propidium iodide (nuclear envelope integrity stain, red); Hoechst 33342 (cell permeant DNA stain, blue); peanut agglutinin lectin conjugated to Alexa Fluor 647 (outer acrosomal membrane integrity stain, cyan). The image gallery also contains two brightfield channels and a darkfield channel (side scatter).