Characterization of equine sperm attributes and selection for intracytoplasmic sperm injection
Date
2018
Authors
Gonzalez-Castro, Raul A., author
Carnevale, Elaine, advisor
Graham, James, advisor
Seidel, George, committee member
Gerrit, Bouma, committee member
Ann, Hess, committee member
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Volume Title
Abstract
When performing intracytoplasmic sperm injection (ICSI), many in vivo mechanisms for sperm selection are bypassed; however, sperm must still be capable of activating the oocyte for successful fertilization. Limited information is available for horses on the effect of sperm preparation method and sperm characteristics that affect ICSI outcome. The overall objectives of this dissertation were to: 1) study the association between sperm sorting methods, sperm population characteristics, and equine ICSI outcome, and 2) characterize sperm oocyte activating factors in stallion sperm, such as phospholipase C zeta (PLCz) and postacrosomal WW binding protein (PAWP). In Experiment 1, a microfluidic device was used to sort frozen-thawed sperm from stallions (n=19), which resulted in a sperm subpopulation with improved motility, morphology, viability and DNA integrity (P<0.05) compared to the original sample. Then, microfluidic sorting was compared with the swim-up procedure and density gradient centrifugation. Swim-up was the least effective method to separate equine sperm. Microfluidic sorting and density gradient centrifugation sorted a sperm subpopulation with similar parameters, improving motility, viability and DNA integrity. After ICSI (n=45), no differences (P>0.3) were observed for cleavage and embryo development among sorting methods. In Experiment 2, sperm population parameters from which individual sperm were selected for injection were analyzed immediately after ICSI and correlated with the outcome. Sperm morphology, viability, membrane integrity measurement of hypoosmotic swelling and DNA integrity were evaluated in frozen-thawed sperm (n=114) used for ICSI in a program. Among sperm parameters, viability correlated positively with normal morphology and membrane integrity (P<0.05). Normal sperm morphology and DNA integrity were not predictive of ICSI outcome. Viability was predictive of cleavage and blastocyst formation, and membrane integrity was predictive of early pregnancy (P<0.05). In Experiment 3, PLCz and PAWP were identified, localized and quantified in stallion sperm, and the relationship with other sperm parameters was investigated. PLCz was identified as a 71 kDa protein and located in the acrosomal and postacrosomal region, midpiece and principal piece of the tail. PAWP was identified by two bands of ~28 and ~32 kDa, located in the postacrosomal region, midpiece and principal piece of the tail. The expression of PLCz and PAWP correlated positively (P=0.04) when analyzed for sperm of 14 stallions. Flow cytometric assessment was feasible for PLCz, but not for PAWP. Expression and percentages of positive labeled sperm for PLCz varied among stallions (n=21). Expression of PLCz was higher in live than dead sperm (P<0.005), and DNA fragmentation correlated negatively with PLCz expression (P<0.04). In conclusion, microfluidic sorting and density gradient centrifugation resulted in a subpopulation of sperm with high quality parameters for ICSI. The probability of sperm-injected oocytes to develop into an embryo and to establish pregnancy improved when sperm were selected from a sample population with higher viability and membrane integrity. This is the first report that describes PAWP in equine sperm, which displayed a novel localization in the midpiece and principal piece of sperm tail in addition to the expected postacrosomal region. Protein levels of PLCz and PAWP were correlated in sperm heads. The expression of PLCz in sperm varied widely among stallions and was associated with DNA integrity. Sperm membrane integrity is indicative of well-maintained plasma membrane architecture, conserving sperm quality and membrane components that are required for oocyte activation and early embryo development. Assessment of PLCz in stallion sperm represents a potent feature to investigate sperm quality for equine ICSI, and potentially can serve as a prognostic biomarker for oocyte activation ability and male infertility. Further studies are needed to determine the relationship between PLCz and PAWP with fertility in horses.
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Subject
microfluidics
PLC zeta
stallion
PAWP
ICSI
sperm