INTRODUCTION:
Semen cryopreservation is today’s leading
technique for banking of male germplasm. However, cryopreservation has detrimental
effects on sperm function and fertility. Sperm
motility is essential to reach and penetrate the egg zona pellucida, and thus
this has been considered one of the most important factors in determining
fertilization rates.
AIM:
Understanding the mechanism of sperm
motility lost due to cryopreservation.
METHODS:
Capacitation was induced in washed/swim-up
bovine sperm (SION Artificial Insemination Centre) incubated in specific
capacitation media.
Images were acquired using an upright
epifluorescence microscope.
Semen motility was analyzed using a CASA device with IVOS software.
For immunoblotting, a western blot of sperm proteins was performed
using monoclonal anti-phospho-PKA Supstrate antibody and HRP-linked secondary
anti-rabbit antibody.
RESULTS:
Bicarbonate deficient capacitation media showed significant
reduction in cryopreserved sperm motility, while no effect on fresh semen.
Glucose addition restored motility losses in HCO₃⁻-deficient media or when mitochondrial respiration was blocked by antimycin
A.
Moreover, when mitochondrial respiration was blocked in the
presence of glucose, inhibition of PKA by H89 did not alter motility.
Additionally,
Western Blot analysis showed positive
correlation between mito-PKA dependent phosphorylation of ≈ 60kDal proteins and
progressive motility.
CONCLUSION:
1.
Bicarbonate has significant enhancing effect on motility of cryopreserved
sperm, indicating
that the efficiency of Krebs cycle to produce CO₂ is relatively
low.
2.
There is insufficient amount of mitochondrial ATP production in cryopreserved
cells.
3. Sperm motility is regulated by mitochondrial PKA.
4. Mitochondrial PKA
mediated phosphorylation of ≈ 60kDa proteins which are associated with ATP-dependent motility.
