Proteomic Markers of Functional Sperm Population in Bovines: Comparison of Low- and High-Density Spermatozoa Following Cryopreservation

Mammalian
semen contains a heterogeneous population of sperm cells.
This heterogeneity results from variability in the complex processes
of cell differentiation in the testis, biochemical modifications undergone
by spermatozoa during transit along the male reproductive tract, interactions
with secretions from accessory sex glands at ejaculation, and, in
the context of reproductive technologies, in the ability of ejaculated
spermatozoa to resist damage associated with freeze–thaw procedures.
When submitted to density gradient centrifugation, ejaculated spermatozoa
distribute themselves into two distinct populations: a low-density
population characterized by low motility parameters, and a high-density
population with high motility characteristics. To understand the origin
of ejaculated spermatozoa heterogeneity, cryopreserved semen samples
from bulls used by the artificial insemination (A.I.) industry were
submitted to Percoll gradient centrifugation. Proteins from low and
high density spermatozoa were then extracted with sodium deoxycholate
and submitted to proteomic analysis using iTRAQ (isobaric tag for
relative and absolute quantitation) methodologies. Quantification
of selected sperm proteins was confirmed by multiple reaction monitoring
(MRM). Overall, 31 different proteins were more abundant in low-density
spermatozoa, while 80 different proteins were more abundant in the
high-density subpopulation. Proteins enriched in high-density spermatozoa
were markers of sperm functionality such as the glycolytic process,
binding to the egg zona pellucida, and motility. Low-density spermatozoa
were not solely characterized by loss of proteins and their associated
functions. Chaperonin-containing TCP1s and chaperones are hallmarks
of the low-density subpopulation. iTRAQ analysis revealed that other
proteins such as binder of sperm proteins, histone, GPX5, ELSPBP1,
and clusterin are overexpressed in low-density spermatozoa suggesting
that these proteins represent defects occurring at different steps
during the sperm journey. These differences contribute to the sperm
cell heterogeneity present in mammalian semen.